BIOLOGY 204/205 Advanced Genetics Laboratory

TABLE OF CONTENTS

Introduction……………………………………………………………………..………….…p.2MODULE 1: Recombinant DNA…………………………………………...…….…..p.9MODULE 2: RNA Interference………………………………………………………..p.22MODULE 3: Proteomics………………………………………………………………….p.34Appendix A: Solutions Guide……………..…………………………………..……...p.42Appendix B: Sterile Technique………………………………………………….......p.54 Appendix C: Spread Plate Technique…………………………………………...…p.55Appendix D: Pipette Use……………………………………………………………......p.55Appendix E: Pipette Exercises …..…………………...….…………………….......p.57Appendix F: GST Plasmid Map………………………………………………………..p.58Appendix G: Plasmid Map …………...………………………..……………….....…p.59Appendix H: DNA/Protein Markers………………………..………………......…p.60Appendix I: Streak Plate Method…………………………………………………...p.61Appendix J: PCR Reagents and Conditions for 1.17……………..……..….p.62Appendix K: Sucrose Gradient Calculations …………………………….…....p.63Appendix L: Sucrose Gradient Tubes after Ultracentrifugation ……..p.64Appendix M: Protein Gel Plate Setup ….…………………………………………p.65Appendix N: Pierce Protein Assay for Module 3 ………………………......p.66Appendix O: Protein Gel Running Setup …………………………….……….…p.67Appendix P: RPM to G-force Conversions………………………….…….……..p.68

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BIOLOGY 204/5Advanced Genetics Laboratory I and II

--- Introduction ---

Module 1 Recombinant DNA/Bacterial Transformation

This module gives you some of the experience you would receive if you were to sub-clone a gene as a part of your research. That is, once you transform a bacterial line with the plasmid that you isolate, you will need to demonstrate that you have made the transfer of the correct gene.

Goals:1. To purify a plasmid and transform E. coli with the plasmid.2. To demonstrate that the transformants carry the plasmid by characterizing the

transformants’ phenotypes. 3. Analyzing the size of the DNA plasmid in a cracking gel. 4. Hybridization with the original plasmid in a Southern blot.5. Amplify the gene inserted into the plasmid by PCR.6. Sequence part of the plasmid.

Module 2 RNA Interference

In this module, a specific target gene product of Paramecium tetraurelia is depleted using an RNAi feeding method. You will isolate RNA from the paramecia and determine if there has been down regulation of the gene product in the RNAi treated population compared to the control.

Goals:1. Design RNAi plasmid and primers2. Test primers for amplification of endogenous mRNA (cDNA) by PCR3. Isolate RNA from harvested paramecia cells4. Synthesize cDNA from the mRNA collected5. Determine the level of target endogenous mRNA by semi-quantitative Reverse

Transcriptase- Polymerase Chain Reaction (RT-PCR)

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Module 3 Proteomics

In this module you will compare the proteins found in wild type and mutant paramecia cilia. You will run a one dimensional polyacrylamide gel, cut out bands of interest, and compare the proteins present in those bands using mass spec.

Goals:1. Use ultracentrifugation to isolate ciliary membranes from wild type and mutant

cells2. Conduct a Pierce Protein Assay to determine the concentration of cilia proteins

in samples provided to you.3. Using SDS-PAGE, run a one dimensional gel and stain with Coomassie blue to

visualize differences in protein content of cilia types. 4. Cut out bands of interest from gel and prepare samples for mass spectrometry

by trypsinizing proteins5. Analyze results from mass spec

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Biology 204/205 Advanced Genetics Laboratory Grading Policy

Biology 204 and Bio 205 are four credit courses. You will complete Modules 1 & 2 and a grant writing exercise during the fall semester for Bio 204. For Bio 205 in the spring semester, you will complete two modules and give a group presentation on the results.

The emphasis of the course is on experimental design, techniques, data gathering and analysis. Work at the bench is given priority over work in a lecture setting. The modules are designed to approach real situations in ongoing research projects. Therefore, the modules are not necessarily designed to be finished in three hours. A few labs will run long, taking 5-6 hours to finish. A few labs will be relatively short. Students are expected to return to lab outside of the scheduled class time, usually at their own convenience, to perform a short manipulation. Sometimes an experiment does not work and it has to be repeated. Coming to class well prepared and following directions carefully will cut down on potential mistakes!

Grading

Your grade will be based on the following components, each with approximately equal weight:

1. Performance in laboratory2. Discussion of experiments in class3. Notebook (these will be checked weekly) 4. Laboratory report 5. Lab meeting presentations6. Oral Exam7. 10% will be deducted from grade per day for late assignments.8. Presentations must be in by 4pm on the assigned date. 10% will be deducted

from grade for late Presentations.

Note: It is important to read over the procedures in the laboratory manual before coming to class. Be prepared to start work after an introduction by the instructor or TA. Check the laboratory calendar so that you know when each module will occur.

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Performance in Laboratory Guidelines

1. Attendancea. Includes arriving on time and staying until work is completeb. Coming in willingly on “off” days when necessary

2. Working well and cooperatively with lab partner(s) a. Simulates behavior in collaborative research groups behavior b. Is behavior disruptive or helpful, distracting, professional?

3. Reading the lab manual in advance and arriving prepared for the day’s methodsa. Working efficientlyb. Following throughc. Being engaged, even when not physically performing experiments

4. "Lab Citizenship"a. Such as following the safety rules, cleaning up, labeling properly, putting

materials away, etc5. Attitude and willingness to participate in experiments

Laboratory Notebook Guidelines

Hard cover, bound notebook; no loose-leaf Record in blue or black ink Number all pages Date all entries Name, course number and email address should be on front cover Reserve 3 pages at the beginning for the table of contents; keep up to date Mistakes should be crossed out with a single line through the entry then initialed Do not skip pages, do not rip pages out Unused portions of a page should have a diagonal line drawn through the blank

portion Each experiment should begin on a new page All data, calculations and graphs should be entered directly into the notebook Neat, orderly, complete

Your notebook should provide enough detail so that another Advanced Genetics student could pick it up and repeat your procedure by following your entries. You should include all of the following information: What was done and why, who suggested it, who did it and when it was done, what results were obtained and what conclusions were drawn.

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Laboratory Report Guidelines

You will be asked to write a formal report of one of the results from one of the lab modules. You will prepare this report as you would prepare a manuscript for publication, with introduction, methods, results, and discussion sections. To aid your preparation of this report, you should go to the library early in the semester and find a short article from Genetics from the last 5 years (download a pdf version or photocopy from a paper journal, the library has both formats). The format in Genetics is appropriate for your report. Below is a description of the content and length of each section.

The report in its entirety should not exceed 10 pages in length. It should be printed double-spaced, with no less than 1-inch margins. It must be in 12-point size in a common font. Each section except the introduction should be started by its section name, in bold type. At the head of the report, you should provide a title that indicates which exercise you are writing about and your name. Whenever possible, you should strive to write succinctly and in the active voice.

Abstract: 250 words summarizing the experiment.

Introduction: The introduction provides an overview of what the report is about, including why the exercise was done (the goal of the exercise) and an explicit statement of the hypothesis or hypotheses being tested. Background information about the biology underlying the exercise should be included in the introduction. Recommended length: 1.5 pages.

Methods: The methods section must be detailed enough to allow the reader to repeat the exercise. You do not need to repeat the detailed description of the protocols in the laboratory manual, but you should refer to the methods in the manual (Format: Laboratory manual Page x-y) at the appropriate points. Recommended length: 3 pages.

Results: The results section reports upon what happened during the exercise. You must include photocopies of the final gels and provide in tabular form other measurements and data you collected. Each figure should have a brief descriptive caption, and each table should have a title. However, it is not sufficient to simply insert these figures and tables. You must interpret your results in the text of the section, with references to the appropriate figure or table (Format: Fig. 1, Table 2A). Recommended length: 2 pages.

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Discussion: In the discussion, you should briefly re-introduce the main goal or hypothesis presented in the introduction, and then describe how your results are related to the goal or hypothesis. In subsequent paragraphs, you should discuss any failures to obtain results, and describe what you believe happened and what you would do differently to correct each problem. This is your opportunity to show how well you understand the molecular processes underlying the protocols! Recommended length: 2 pages.

Safety in the Laboratory

General Rules:1. Disinfect your bench top with a 10% bleach solution when you arrive and when

you finish lab.2. Wear gloves and avoid touching face and/or hair during an experiment. 3. Wash your hands before you leave lab.4. Do not eat, drink, smoke, chew gum or apply cosmetics while in lab.5. Dispose of all used materials as directed.6. Keep aisles clear.7. Wipe all spills immediately. Inform lab tech and/or TA if you spill ANYTHING.8. Dispose broken glass in the appropriate receptacle. Inform lab tech and/or TA

that you have broken glass.9. Wear closed-toed shoes.10. Tie back long hair.11. Try to avoid wearing baggy, loose clothing that can interfere with your

experiment and may catch on fire. 12. Extinguish burners as soon as you finish using them.13. All Chemical Safety and MSDS information is located in the binder on the back of

the door.14. If you are unsure about a procedure, just ask.

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Pipetting

1. Acquaint yourself with the various denominations of pipettors in an attempt to avoid mistakes, particularly when working under time pressure.

2. The height of the fluid in the glass pipettes is measured at the bottom of the meniscus while the pipette is being held vertically.

3. Never put a pipette back into a sterile container.

4. Do not handle the lower part of the pipette.

Serial Dilutions: Serial dilutions allow you to dilute a sample many fold by making a series of small dilutions.

Standard Dilution Steps: Unless special circumstances demand it, the following are the only dilution steps that are used (For convenience and error avoidance in performing the accompanying arithmetic): 10, 20, 50 and 100.

10X 1:10 0.1 ml/0.9 ml 100 µL/900 µL

20X 1:20 0.1 ml/1.9 ml 50 µL/950 µL

50X 1:50 0.1 ml/4.9 ml 20 µL/980 µL

100X 1:100 0.1 ml/9.9 ml 10 µL/99 0µL

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Module 1 Recombinant DNA

Please refer to page 2 for introduction

**Note: The E.coli cell line used in this module is K12. The K12 bacteria cells are to be transformed with the GST plasmid.

1.0 Overnight (ON) Bacterial Culture (Done for you) 1. The lab tech will add 2.5 ml of cells previously grown ON to 125 ml LB amp

medium (per group).2. The cells will grow with shaking at 37C ON.

1.1 Isolation of Plasmid DNA HAZARDOUS CHEMICAL INFO: -Salt-Saturated Phenol is to be used only while wearing gloves under the hood. Dispose of all pipets and liquid waste containing SS Phenol in appropriate disposals. -Chloroform: Isoamyl Alcohol (24:1) is to be used only while wearing gloves under the hood. Dispose of all pipets and liquid waste containing C:IA in appropriate disposals.

1. Transfer 125ml of the overnight bacterial culture (Transformed E. coli) to a large, sterile, screw top centrifuge bottle and harvest the bacteria by centrifuging at 5000 rpm, 4°C for 10 minutes in the Beckman J2-21.

2. Decant the supernatant broth into the waste jar.3. Resuspend the bacterial pellet in 5ml of Solution I containing 5mg/ml lysozyme.4. Transfer to a 30ml polycarbonate screw top Oakridge centrifuge tube. Let stand

at room temperature for 5 minutes.5. Add 10ml of freshly made Solution II. Place the cap on the tube and mix the

contents by inverting the tube several times. Mix gently. Let stand on ice for 10 minutes.

6. Add 8ml of ice-cold 5M potassium acetate (pH 4.8). Fill tubes only ¾ full. Screw on the cap and mix by inverting. Let stand on ice for 30 minutes.

7. Balance the tubes before centrifugation.8. Centrifuge in the Beckman J2-21; 15,000 rpm, 4°C for 20 minutes. The genomic

DNA and bacterial debris should form a tight pellet at the bottom of the tube. 9. Being very careful not to disturb the pellet, use a 10ml pipet and transfer all to a

30ml glass screw top tube. Only take clear supernatant. 10. Add at least 500µL of heat treated RNase A to each tube. Please use all of the

RNase A provided. 11. Incubate at 37ºC for 30 minutes in Innova 4000.

12. In the chemical flow hood, add one volume of SS (salt saturated) phenol using glass pipettes. (Note the yellow color which helps you identify the phenol phase

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in the next step.) Your tubes can be no more than 2/3 full including the addition of the phenol, so divide your original solution as necessary into 3 or 4 conical tubes, using glass pipettes. SAFETY NOTE: Phenol can cause severe burns to skin and damage clothing. Gloves, safety glasses, and a lab coat should be worn when working with phenol. All manipulations should be carried out in a fume hood. A glass receptacle is available exclusively for disposing of used phenol and chloroform.

13. Vortex the conical tube and contents with lids on for 1 minute; be sure the contents are thoroughly mixed. Make sure the tops of the conical tubes are screwed on tightly to ensure that no leaking will occur. Centrifuge for 1 minute at 2800 rpm using the Eppendorf Centrifuge 5702.

14. Transfer the non-colored upper, aqueous phase to a fresh conical tube. Do not take the interface which is denatured protein. In the hood, add 1 volume of chloroform: isoamyl alcohol (24:1) – the same amount as the phenol you added. Vortex 1 minute and centrifuge 1 minute at 2800 rpm.

15. Transfer the upper, aqueous layer to a fresh 30ml glass tube and add 2.5 volume of cold 95% ethanol, using glass pipettes. You need to calculate how much total liquid will be in each 30ml glass tube. The tube cannot be more than 2/3 full, so you may have to use more than one 30ml glass tube. Make your calculations before adding the ethanol!

16. Mix and allow it to precipitate in the -80o freezer overnight.Next Day

17. Balance your tubes along with their rubber sleeves.18. Recover the DNA by centrifuging the tube at 4°C in the Beckman J2-21 at 9500

rpm for 30 minutes. 19. Discard the supernatant into a waste container. The pellet will look like a whitish

residue on the side of the tube. 20. Resuspend the pellet with 1 ml 70% ethanol by pipetting up and down onto the

sides of the tube. Try to resuspend the entire pellet to increase your plasmid yield. Transfer the resuspention from the first tube to the next tube until all pellets are resuspended and pooled together. Transfer the solution into one sterile 1.5ml microfuge tube.

21. Microcentrifuge for 5 minutes at 14,000 rpm. Pipet out the ethanol; add 1ml more of 70% ethanol to wash the pellet and vortex for 30 seconds. Spin at 14,000 rpm for 5 minutes.

22. Discard the ethanol; dry the pellet using the SpeedVac in the basement. Give your sample to the TA/Lab tech to be properly dried for 15 – 20 minutes.

23. Dissolve the pellet in 0.3ml TE. Aliquot 100 L to each of 3 microcentrifuge tubes (properly labeled!).

24. Store at -20°C.

1. 2 Agarose gel to confirm isolation of the plasmid HAZARDOUS CHEMICAL INFO:

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-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.-UV light is very harmful if looked at directly. When viewing your gels on the UV light box be sure to wear a protective face mask, or place the shield on top of the box before turning on light.

1. Prepare 300ml 1X TAE from 5X TAE stock.2. Dissolve 0.35g agarose in 50ml 1X TAE buffer to make a 0.7% gel.3. Microwave on high for 1 minute.4. Swirl the flask and make sure all of the agarose is dissolved. If not, microwave

until it is. Remove flask with a hot mitt. 5. Place the running tray into the gel-casting tray. Add comb. 6. Cool agarose slightly; approximately 5 – 10 minutes, swirling occasionally. Slowly

pour agarose into the farthest corner from the comb in the gel casting set up. Try to avoid bubbles! If bubbles appear remove them with a pipette tip. Let cool until opaque (approximately 20 minutes).

7. While your gel is setting, thaw out one tube of your plasmid DNA on ice. Just before you are ready to load the gel, heat the λ Hind III marker for 7 minutes in the 65°C hot block. – PLACE ON ICE IMMEDIATELY.

8. Mix 4µL of 6X DNA sample buffer with 20µL plasmid DNA on a piece of Parafilm.9. Once your gel is set, remove it from the casting tray. Place it in the running tray,

with the comb still set. Cover the gel with 1X TAE. Gently remove the comb. Removing the comb last will ensure that your wells do not collapse.

10. Be prepared to load the gel quickly—you do not want your DNA to diffuse into the running buffer.

11. Load 24µL of plasmid DNA sample and 20µL of λ Hind III marker in the wells; put the lid on the box so that the DNA will run toward the red electrode.

12. Run the gel at 100V for ~1 hour.13. Stain the gel for approximately 15 minutes in ethidium bromide, and destain in

water for 5 minutes.14. Examine the gel on the UV light box. If the ladder is not visible or is faint, place

the gel back into the stain. When you feel that your gel is properly stained, take a picture to document your results.

15. Leave the gel in destain to be discarded later. 16. Rinse electrophoresis unit with RO water after use so the buffer does not dry on

the electrodes.1.3 Grow an overnight broth culture of E. coli ( Done for you)

1.4 Transformation Three hours before class the tech will take 1 ml of an ON culture and inoculate 50 ml of fresh LB broth with it. It will shake at 37C for two hours. This will produce cells in exponential growth phase for you to transform.

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1. Divide broth culture into 2 sterile 30ml screw top Oakridge centrifuge tubes; place tubes in ice for 30 minutes.

2. Thaw out one tube of your plasmid DNA on ice.3. Centrifuge the cultures at 4°C in the Beckman J2-21 for 10 minutes at 5000 rpm;

decant the supernatant into the collection flask provided.4. Resuspend one pellet in 25 ml ice cold 50 mM CaCl2. Combine this

resuspension solution with the second bacterial pellet; place on ice for 20 minutes. Keep CaCl2 on ice while waiting.

5. Centrifuge the cell suspension at 4°C in the Beckman J2-21 for 10 minutes at 5000 rpm.

6. Decant the supernatant and resuspend gently the pellet in 3ml ice-cold 50 mM CaCl2; place on ice for 5 minutes.

7. Dispense 2 aliquots of 0.3 ml cells in ice-cold 1.5ml microfuge tubes; add 0.2 ml of transformation buffer to each tube. Save remaining competent cells at 4°C.

8. Add 5 µL plasmid DNA to one tube. The second tube will not contain plasmid DNA and will act as a control. Mix gently and leave on ice for 20 minutes.

9. Heat shock cells for 1 min in 42°C water bath.10. Plunge tubes into ice and let sit on ice for 5 minutes. 11. Add 0.7 ml LB to each tube and tap gently with finger.12. Shake at 37°C for 60 minutes in Innova 4000.

**NOTE: during this hour incubation your TA or Lab Tech will demonstrate proper spreading and streaking procedures for plating. It is very important that you understand sterile technique when working with bacteria so you don’t contaminate your samples.

13. Plate 0.05, 0.1, and 0.3 ml of the cells with plasmid DNA onto LB amp plates. Use the spread plate technique. Let the plates dry for 5 minutes right-side up before inverting and placing in the incubator.

14. Streak (Do not use the spread plate technique) the contents of the “no DNA tube” on an LB amp plate and an LB plate. The LB amp plate will act as a negative control, while the LB plate will serve as a positive control.

15. Label plates appropriately with group number, date, type of bacteria, and any other important information, such as how much bacteria was plated.

16. Incubate the plates at 37°C overnight (upside down); be sure to remove, wrap in Parafilm and refrigerate the plates tomorrow!

Following Day: 1.5 Selecting for bacteria that carry the plasmid___1. Examine transformed and no DNA control plates. (There should be no colonies

on the “No DNA” LB amp plate).2. Choose 6 well isolated colonies from the transformed plates. Streak each colony

on half of an LB amp plate. 3. Choose 2 well isolated colonies from the control (non-transformed) plate

provided. Streak each colony on one half of an LB plate.

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4. Incubate the plates overnight at 37°C.5. Wrap the old plates in Parafilm and refrigerate.

1.6_Secondary selection of transformed bacteria_________________ 1. Transfer 4 well-isolated colonies from 4 different transformed streaks and 2

control colonies into separate 1ml aliquots of sterile saline. Refrigerate the old plates.

2. For the transformed bacteria, streak 1 loopful of saline/bacteria suspension onto ½ of an LB amp plate. Do this for each of the 4 samples.

3. For the control cells, streak 1 loopful of the saline/bacteria suspension onto ½ of an LB plate. Be sure to label plates clearly!

4. Incubate at 37°C overnight; remove and refrigerate the next day.5. Go to 1.16

1.7 Preparing bacteria for the cracking gel (day before 1.8) 1. Using a marker, draw a line down the center of a new LB amp plate. Make a

template on paper with 1.5 cm x 1.5 cm squares on each half. Place the plate over the template.

2. Using sterile tweezers, select a sterile toothpick.3. Choose 2 LB amp plates from Day 1.6 that show the best growth. With the

toothpick, select one colony from the Day 1.6 plate and “fill in” the square on the agar on the plate. Repeat for the 2nd colony using a new toothpick.

4. Repeat the procedure for the control, but use a fresh LB plate.5. Incubate at 37°C overnight for at least 24 hrs, but less than 36 hrs.

1. 8 Next day: Cracking gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300ml 1X TAE.2. Prepare 0.7% agarose gel.3. Use a sterile toothpick to scrape bacteria from the plates prepared the day

before. Add bacteria from each square to 250µL of cracking buffer (Two squares for one tube of 250µL of cracking buffer). Do this for transformed and non-transformed cells (you should have a total of 2 microcentrifuge tubes). Vortex tubes to mix well.

4. Incubate at 37°C in the hot water bath for 25 minutes.5. Centrifuge for 15 minutes at 14,000 rpm.6. Use a toothpick to remove the bacterial debris from the bottom of each tube.

(You won’t be able to see a pellet, but when you pull it out, it will look like a blue glob).

7. Load the gel slowly and carefully:

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Lane 1: 20µL Hind III marker (Heat in 65C hot block for 7 minutes before loading) Lane 2: 20µL plasmid DNA solution (10µL plasmid DNA + 4µL 6X DNA sample buffer + 6µL 1X TAE)Lane 3: Transformed supernatant Lane 4: 50µL Cracking buffer only Lane 5: Non-transformed supernatant Note: Load as much transformed and nontransformed supernatant as possible (A well-formed well can hold ~50 L).

8. Run the gel for 1 hour at 100 volts.9. Stain with ethidium bromide, destain, and photograph. Look for genomic DNA,

plasmid DNA and RNA.

1.9 Labeling DNA with Biotin -Salt-Saturated Phenol is to be used only wearing gloves under the hood. Dispose of all pipets and liquid waste containing SS Phenol in appropriate disposals. -Chloroform is to be used only wearing gloves under the hood. Dispose of all pipets and liquid waste containing chloroform in appropriate disposals.

Part A: Labeling Reaction1. Remove an aliquot of Plasmid DNA from the refrigerator and place on ice. 2. Add labeling reaction components to a 0.5ml tube (on ice):

dH2O 128µLdNTP mix 28µL1X DNase I Buffer 19.9µLDNase I 0.1µLPlasmid DNA 4µLDNA Polymerase I 20µL

3. Mix well and centrifuge for 5 seconds at desktop spinner.4. Allocate 50µL into 4 tubes.5. Incubate at 15°C for 2 hours in thermocycler.6. Add 5µL Stop Buffer to each tube and mix.7. Incubate tubes at 65°C for 5 minutes in thermocycler.

Part B: Purification of DNA probes1. Transfer liquid to consolidate solution from 4 tubes into one tube.2. Add 4µL 10% SDS to tube and mix.3. Add 110µL Chloroform and 110µL SS Phenol to an empty 1.5ml

microcentrifuge tube.4. Transfer DNA solution to chloroform phenol tube. Vortex 2 minutes and

then centrifuge for 2 minutes at 14,000 rpm5. Collect the top layer of liquid and transfer to a fresh 1.5ml tube. Discard

remaining liquid into waste container.

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6. Add 220µL chloroform to tube. Vortex 2 minutes and then centrifuge for 2 minutes at 14,000 rpm.

7. Collect top liquid layer and transfer to a clean tube. Discard remaining liquid into waste container.

8. Add 40µL 3M Sodium Acetate (pH 4.8) and 800µL cold 95% ethanol. Mix gently by inverting tube.

9. Store at -20°C ON (at least 6 hours)The Next Day:

10. Centrifuge for 5 minutes at 14,000 rpm.11. Carefully remove the supernatant.12. Resuspend the pellet in 1ml cold 70% ethanol. Centrifuge for 5 minutes at

14,000 rpm. (1st Wash)13. Remove supernatant (ethanol). 14. Resuspend the pellet in 1ml cold 70% ethanol. Centrifuge for 5 minutes at

14,000 rpm. (2nd wash)15. Remove supernatant (ethanol). Let tube dry on lab bench for at least 1 hour. 16. Once dry, resuspend probe in 12µL TE buffer and store at -20° C.

1.10 Preparing for the Southern Blot (day before 1.11) 1. Using a marker, draw a line down the center of the underside of a fresh LB amp

plate. Draw two 1.5 cm x 1.5 cm squares on the underside of the plate, one on each half.

2. Using a sterile toothpick, pick one isolated colony from the Day 1.6 LB amp transformed plate. “Fill in” one square on the fresh LB amp plate with one colony. Repeat for the second square making sure to use a fresh toothpick.

3. Repeat steps one and two, this time using an LB plate and the Day 1.6 non-transformed cells.

4. Incubate both plates for at least 24 hours.

1. 11 Southern Blot HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Run cracking gel (same as Day 1.8, except add 20ul of plasmid mixed with 4ul 6X DNA sample buffer to the plasmid lane). Do not forget control lane!

2. Stain with ethidium bromide, briefly destain, and examine the gel. 3. Make sure to destain the gel for approximately 5 minutes before denaturing.

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4. Photograph the gel before destaining completely—you will use this photograph later to compare to the results of your southern blot.

5. Denature gel in 0.5 M NaOH/0.8 M NaCl for 30 minutes, rocking. Decant the solution and repeat.

6. Rinse gel in dH2O for 1 minute.7. While the gel is rinsing, cut and hydrate the nitrocellulose filter for 3 minutes in

dH2O, then in 10X SSC until blot set-up is ready. Make sure to notch the corner of the nitrocellulose for orientation purposes and always wear gloves when handling the nitrocellulose. Always handle the filter with forceps, and only around the edges so as to not create blotches of background color.

8. Neutralize gel in 0.5 M Tris/1.5 M NaCl (pH 7.0) for 30 minutes, rocking. Decant the solution and repeat.

9. Rinse the gel in 10X SSC for 3 minutes, rocking.10. While the gel is neutralizing, prepare the Test Spot.

a. Take your Biotin labeled probe out of the freezer and let thaw on ice.

b. Cut a piece of nitrocellulose approximately 1 cm x 1 cm. Make sure to cut your test spot in a unique way so that you can identify it later. For example you can cut one or two small notches on the side of the square or cut off a corner.

c. Hydrate the test spot in dH2O for 3 minutes.d. Soak the nitrocellulose in 10X SSC until the probe is thawed. e. Remove the nitrocellulose from the SSC and place on a small

Kimwipe.f. Add 2 µL of probe to the center of the square of nitrocellulose.g. Let dry on Kimwipe, then wrap in plastic wrap and store in the

freezer until 1.12.11. Assembling the Southern Blot:

-First the wick (a long strip of paper towel will work) needs to be placed on the platform so that it can only touch the buffer on two sides.

-Place three pieces of Whatman 3M filter paper on top of wick. -The gel should be placed on top of the filter paper and the nitrocellulose

on top of that. **Make sure the nitrocellulose and the gel are lined up in the correct orientation so you can compare them later**

-Place three more pieces of Whatman 3M filter paper on top of the nitrocellulose.

-A stack of cut paper towels at least 10 cm high should be assembled and placed on top of the filter paper (All filter paper and paper towel should be cut to the size of the gel).

-Wrap the whole set up in plastic wrap to provide stability to the stack and minimize evaporation.

-Pressure should be applied to the top of the stack to enhance wicking overnight. (Your TA should demonstrate this and assist in the assembly)

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12. Let Southern Blot transfer ON in 10X SSC.

1.12 Drying of Blot (Done for you)______________________________1. Disassemble the Southern blot and rinse the nitrocellulose in 5X SSC for 2

minutes.2. Dry on large Kimwipe.3. Bake nitrocellulose blot on a kimwipe and test spot wrapped in plastic in vacuum

oven at 80°C for 2 hours.4. Carefully place blot into hybridization bag and seal on all four sides. 5. Store blot and test spot in freezer.

1.13 Hybridization of the Southern Blot 1. Carefully unwrap your test spot. 2. Place your uniquely cut test spot in a small plastic tub containing all of the test

spots from the class. Your TA or lab tech will hydrate these in 2X SSC and then place them in prehybridization solution and return them to you tomorrow for 1.14.

3. Cut a corner of your hybridization bag. Using a stereological pipette, add 50ml 2XSSC to the bag to hydrate your blot. Reseal the corner of the bag using the food sealer. The blot should be uniformly hydrated after several minutes.

4. While the blot hydrates, denature 200L of Herring sperm DNA (2mg/mL) in 100°C hot block for 10 minutes followed by plunging into ice water.

5. For prehybridization of the nitrocellulose blot, add the 200µL of freshly denatured Herring sperm to the prehybridization solution and mix.

6. Cut a corner of your bag and remove the 2X SSC. Pour in the prehybridization solution using a stereological pipette. Reseal the corner making sure to push all of the air bubbles out of the bag. If there are still more air bubbles in the bag after you have resealed the corner, set the bag upright and push all the bubbles to the top of the bag. Reseal the bag across the top to trap the air bubbles away from the blot.

7. Incubate at 42°C while rocking for 2 hours. The volume of prehybridization solution used should be 20 to 100L per cm2 of the blot.

8. For hybridization, heat-denature 5L of the probe made on 1.9 and 200 L of Herring sperm DNA by placing in 100°C hot block for 10 minutes. While in the heat block, wrap tops of tubes in Parafilm to preserve the label in the next step.

9. Plunge into ethanol ice slurry for fast chilling, making sure not to erase all labels written in marker. Just before use, add to the hybridization solution.

10. Remove the prehybridization solution from the bag by cutting a corner and pouring off. Add the hybridization solution to the bag (20-100 L per cm2) and reseal using the same techniques described in step 6. The blot should be hybridized at 42°C overnight while rocking to achieve maximal sensitivity.

17

Following Day: 1.14 Detection of the DNA **All the washes in this section need to be completed while rocking.**

Decant and save the hybridization solution in an appropriate size tube. Store at 4C.1. Wash the blot & test spot with 100ml of 2X SSC/0.1% (w/v) SDS at room

temperature for 3 minutes. Decant the SSC and repeat.2. Wash the blot & test spot with 100ml of 0.2X SSC/0.1 % (w/v) SDS at room

temperature for 3 minutes. Decant the SSC and repeat.3. Wash the blot & test spot in 100ml of 0.16X SSC/0.1% (w/v) SDS at 50°C for 15

minutes. Decant the SSC and repeat.4. Rinse the blot & test spot in 100ml of 2X SSC at room temperature for 1 minute.5. Dry on large Kimwipe and then wrap in plastic wrap and store in refrigerator.6. The hybridization mixture containing the biotin-labeled probe may be reused.

Store the mixture at 4C for several days or at -20C for longer periods. Placing the hybridization solution in a boiling water bath and cooling on ice just prior to use should denature the probe.

1. 15 Development of Blot HAZARDOUS CHEMICAL INFO:-NBT/BCIP is highly toxic. WEAR GLOVES when handling and dispose of all liquid waste containing NBT/BCIP in the appropriate waste container.

1. Wash the blot and small test square in Buffer 1 at room temperature for 1 minute with sufficient buffer to cover. Decant Buffer 1 into the sink.

2. Incubate blot and test spot in Buffer 2 in a plastic container for 1 hour at 65°C, rocking, with sufficient buffer to cover. Then decant off.

3. Dry blots between two Kimwipes.4. Dry blots at 80o C for 15 mins.5. Wash the blots in freshly made strep-avidin alkaline phosphatase (SA-AP)

conjugate for 25 minutes at room temperature. 1µL SA-AP per 1mL Buffer 2. (Add only enough SA-AP conjugate to cover the blots (~10ml). Use gentle agitation and occasionally pipette SA-AP over the blots.)

6. Decant and save the SA-AP in a 15ml tube. Save for step #8.Wash the blot and test spot in Buffer 1 using 20 to 40-fold greater volume than employed in step 3. Gently agitate blot for 15 minutes in Buffer 1.(if you used 10 ml diluted SA-AP conjugate in step 3, wash with at least 200-400ml Buffer 1.) Decant Buffer 1 into the sink.

7. Wash the blots for 10 minutes in Buffer 3, rocking. Decant Buffer 3 into sink. (Do steps 8 and 9 at the same time and monitor the rate of color development. The tube of saved SA-AP acts as a positive control.)

8. Add 1ml NBT/BCIP solution to the saved SA-AP. A blue color should develop overtime. Wear gloves when working with NBT-BCIP.

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9. Add 9 ml of NBT-BCIP solution to the blots. Allow the blots to develop for 15 minutes to 1 hour. Agitate the Tupperware.

10. DNA bands will be most evident on only one side of the blot (check your notch for correct orientation). Check your blot every 2 minutes to ensure that over-development does not occur.

11. Once bands have developed, decant the NBT-BCIP solution in the appropriate waste container and wash the blot in TE. This will terminate the color development reaction. The TE can then be decanted into the sink.

12. Let the blots dry on a large Kimwipe. Then wrap in plastic wrap and label. The lab tech will photograph and distribute the blots for your notebooks.

13. Measure the photograph of the cracking gel, and compare the relative position of the plasmid band to the results of the blot. Interpret your results.

14. Record the development time with BCIP/NBT

1.16 Designing Primers

5’-------------------------------------------------------------------------------------------------------3’ 698 bp

GST

Partial DNA Sequence for GST (Read left to right, top to bottom):

5’…GTATTCATGTCCCCTATACTAGGTTATTGAAAATTAAGGGCCTTGT 310

GCAACCCACTCGACTTCTTTTGA……….ATCCTCCAAAATCGGATCTGGT 960

TCCGCGTGGATCCCCGGGAATTCATCGTGACTGACTA………….…………..3’

The glutathione S-transferase protein consists of 232 amino acids. The sequence—using the one-letter abbreviation for each amino acid—is shown below.

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPGIHRD

Using this information, design the primers to amplify the GST gene. Once you have designed the primers, fill out the oligonucleotide request form. The primers will then be made on a DNA synthesizer.

1. 17 PCR

1. Set up 7 - 0.5 ml PCR reaction tubes according to the PCR chart in Appendix I. Read the chart carefully and make sure you add the correct amounts of reagents. PCR is a very sensitive reaction and adding the incorrect amounts of reagents

19

may cause poor results. Appropriately label your tubes with your group color and tube number!

2. For Sample 4, pick up three individual colonies from your transformed plate with a sterile toothpick and place into a 1.5 ml microfuge tube filled with 50 µL of sterile dH2O. Take 3µL of this bacterial solution and use as your “template DNA”. Do the same for Sample 5 using non-transformed bacteria.

3. Before mixing the reactants, calculate how much water must be added to make a total of 25L (including the Taq Polymerase). This is necessary because the amount of template DNA that you add might differ from tube to tube.

4. If you add too much DNA, nonspecific amplification may occur—ask your TA how much DNA to add based on the approximate concentration of your plasmid samples.

5. Add all reactants, except the Taq, while the tubes are on ice.6. Lastly add the Taq polymerase.7. Once all reactants are added to the tubes, spin them briefly to bring all the liquid

to the bottom of the tube. Note: Only spin tubes briefly (5 sec.), 0.5 ml tubes are thin-walled and can crack if microfuged for too long.

8. Keep the tubes on ice until the entire class is ready to load the thermocycler.9. The thermocycler will run for approximately 3hrs. After the 3hr. period is over,

the thermocycler will stay at a constant 4C until the tubes can be placed in the refrigerator by the lab technician or TA. This will ensure that the PCR products will not degrade.

1.18 Examining the PCR product HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300 ml 1X TAE.2. Prepare a 2% agarose gel. Note: The 2% agarose solution will solidify quickly!

Pour gel while still relatively hot. 3. Remove 20µL of PCR product from each tube; add to 4 µL of 6X sample buffer.

Store the remaining PCR product at 4°C. 4. Once your gel is set, remove the comb and place the gel in the running box.

Cover the gel with 1X TAE buffer. 5. Load your DNA samples and 10µL of 100bp ladder into the gel. 6. Run gel at 100 volts for 1 hour.7. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium

bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.8. Destain, examine, and photograph gel.

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1.19 Searches of the sequence using BLAST ( B asic L ocal A lignment S earch T ool) The plasmid DNA has been sequenced. You will receive a printout of the results. You will analyze this information using a computer program called BLAST.

To access the program, go to http://www.ncbi.nlm.nih.gov/BLAST

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Module 2RNA Interference

Introduction In this module, a specific target gene product of Paramecium tetraurelia is depleted using an RNAi feeding method. Paramecia are fed with an RNAase III-deficient E. coli strain, HT115 bacteria transformed with a gene specific RNAi construct. The RNAi construct has T7 RNA polymerase promoters on either side of the cloned gene which expresses double stranded RNA upon IPTG induction. After 48 hours of feeding at 28°C the level of target endogenous mRNA is determined by semi-quantitative Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR).

The lab tech has transformed E. coli HT115 cells. The “test” cells contain a plasmid with the PAWN A gene, while the “control” cells contain a plasmid without the PAWN A gene. Both the test and control cells are resistant to ampicillin and tetracycline.

2.1 Design Primers and PCR Protocol___________________________

5’-------------------------------------------------------------------------------------------------------3’641 bp – PAWN A Sequence

ATGTATTTAT TAATTTTAAG TATATTGTAA TTTGGCATCG TGATTTAAGC 50

TCAAGAGACA AACAATACTG AAGAAGAGAT TTCAGATTAT TGTGATGCAG 100

TTGCCAAAGA CACTTCTTTT AAGGTGAATG TAACAGTTTC AGATATTAAC 150

AATAAAAATT ATTGTGTTGA AGGTGGATCT CGTGTGGCTT TATTCGACAC 200

AATTTAACAA GAAGATCAAT ATGTGTATTT GTCTGAACAT TATTGTGCCA 250

ACTTATAACA TTATCCAATT ACTTGCGAAC AATATTATCA CGCTTCAGAA 300

TATGATAAAA AA GTAAGAAT TTAAATACTA TCA ATTAGGC CAAAGCCAAC 350

TATAAGAAGC TCTATGAAGA TTATAAGGCC ACTGGAGTAC CCAACTCTGA 400

TTGTTTGGGT ATTGCAAGAT TCGTTTTCTG TGCTGAATAA TTCAAATATT 450

GCAGCACAGA TGATGGAAAT ACCGATTATG AAATCTGCAG TTTCTTATGT 500

GTCATTTGGC AAAATAGATG TCCTGATTAC AGTGATATTT ACGATCGTGT 550

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TTGTGCTAAT GGAGGAGGAG AAAATGGAAG ATGCAGTTAT GCAATTAACT 600

ATACTTTTCT GTTGTTTTTC ATTCTATTTT TATTATATTG A 641

The intron is nucleotide 313 to 333 and it is highlighted.RNAi Construct is underlined. 560 bp (21 580)

Using this information, design primers and test your primers for endogenous mRNA. Once you have designed the primers, fill out the oligonucleotide request form. The primers will then be made on a DNA synthesizer.

Using the primers you designed, calculate the annealing temperature. Fill out the following tables with the PCR cycle information.

Endogenous control PCR cycle conditionsTemperature (°C) Time (minutes) Cycle # of Cycles Initial Denaturation 1 Denaturation Annealing Extension Final Elongation 1

2.2 Run RNAi and Endogenous PCR____________________________Run the PCR protocols you designed with the primers you designed on genomic Paramecium tetraurelia DNA. Use the following table to set up your PCR.For each primer set you will run four different reactions.

Components Tube 1 Tube 2 Tube 3 Tube 4Template DNA (~100ng) 1 µL 1 µL 1 µL 1 µL10X Buffer 2.5 µL 2.5 µL 2.5 µL 2.5 µL25mM MgCl2 0 µL 1.5 µL 3 µL 4.5 µL10mM dNTPs 0.5 µL 0.5 µL 0.5 µL 0.5 µL20µM F primer 0.5 µL 0.5 µL 0.5 µL 0.5 µL20µM R primer 0.5 µL 0.5 µL 0.5 µL 0.5 µLTaq Polymerase 0.25 µL 0.25 µL 0.25 µL 0.25 µLdH2O 19.75 µL 18.25 µL 16.75 µL 15.25 µLTotal 25 µL 25 µL 25 µL 25 µL

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2.3 Examine PCR product______________________________________ HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300 mL 1X TAE. 2. Prepare a 2% agarose gel using a 12 well comb. Note: The 2% agarose solution

will solidify quickly! Pour gel while still relatively hot. 3. Remove 10 µL of PCR product from each tube; add to 2 µL of 6X sample buffer.

Store the remaining PCR product at 4°C. 4. Once your gel is set, remove the comb and place the gel in the running box.

Cover the gel with 1X TAE buffer. 5. Load your DNA samples and 10 µL of 100bp ladder into the gel. 6. Run gel at 100 volts for 1 hour.7. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium

bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.8. Destain, examine, and photograph gel.

2.4 Optimize PCR Protocol_____________________________________After discussing the results with your instructors, optimize your PCR protocol.

2.5 Agarose Gel of Optimized PCR Protocol______________________ HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300 mL 1X TAE.2. Prepare a 1.5% agarose gel. Note: The 1.5% agarose solution will solidify

quickly! Pour gel while still relatively hot. 3. Remove 20 µL of PCR product from each tube; add to 4 µL of 6X sample buffer.

Store the remaining PCR product at 4°C. 4. Once your gel is set, remove the comb and place the gel in the running box.

Cover the gel with 1X TAE buffer. 5. Load your DNA samples and 10 µL of 100bp ladder into the gel. 6. Run gel at 100 volts for 1 hour.7. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium

bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.8. Destain, examine, and photograph gel.

2.6 ON Bacterial Culture (Done for you)_________________________The lab tech will start ON cultures from freshly transformed test and control HT115 cells. The cells will grow with shaking at 37C overnight.

2.7 Induce Bacteria and Feed to Paramecium_ ____________________

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Part A: Inducing bacteria1. Five hours before lab, the lab tech will add 1 mL of the test and control cells

previously grown ON to 50 mL LB amp medium.2. The cells will shake at 37C for two hours.3. After two hours of shaking measure the OD value of the culture at 595 nm using

the spectrophotometer. Use LB as the Blank. When the OD value reaches between 0.3 and 0.4, proceed to the next step.

4. Add 125 µL 0.2M IPTG to each 50 mL bacterial culture. Shake at 37°C for 3 hours.

Part B: Purging Paramecium Cells(Your TA will demonstrate procedure in MLS 224)

1. Obtain a 100 mL flask of paramecia grown in bacterialized wheat culture from the 28C incubator. (These cells were added to the bacterialized wheat culture 4 days earlier and are now feeling starved.)

2. Pour cells through funnel that has a small folded Kimwipe nestled inside into a 100 mL pear-shaped centrifuge tube

3. Balance the centrifuge tube against a centrifuge tube filled with water and centrifuge at ¾ speed for 2 minutes in the IEC HN-SII centrifuge.

4. Obtain a 15 mL plastic centrifuge tube containing 10 mL Dryl’s solution. 5. After centrifugation, your TA will demonstrate how to transfer the cells from the

bottom of the pear-shaped to the Dryl’s solution using a glass Pasteur pipette. 6. Keep the cells in the Dryl’s solution until you are ready to add them to the flasks

containing your test and control bacterial cultures.Part C: Preparing the bacterial cultures for the paramecia

1. After three hours of shaking, transfer the test and control bacterial cultures to large, sterile centrifuge bottles and harvest the bacteria by centrifuging at 5000 rpm, 4°C for 10 minutes in the Beckman J2-21 (JA-14 rotor).

2. Decant the supernatant broth into the waste jar.3. Resuspend each bacterial pellet in 100 mL wheat culture containing 100 µL Amp,

100 µL Stigmasterol, and 250 µL 0.2M IPTG. Transfer resuspended pellet to flask.4. Transfer approximately 1 mL of paramecia in Dryl’s solution to a depression slide

under a dissecting microscope. Using a Pasture pipette count and transfer 350 paramecia to each flask of culture.

5. Place flasks in 28C incubator.

2.8 Induce paramecium culture (Done for you)___________________After 24 hours of incubation, your TA will add 100 µL Stigmasterol and 250 µL 0.2M IPTG to each of the cultures and continue to incubate at 28C.

2.9 Harvest cells and Isolate RNA

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HAZARDOUS CHEMICAL INFO:β-Mercaptoethanol is toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the 2-Mercaptoethanol) in the appropriate waste container. Do not inhale fumes.

Induce Paramecium culture (Done for you) After 48 hours of incubation, your TA will add 100 µL Stigmasterol and 250 µL 50mM IPTG to each of the cultures and continue to incubate at 28C.

Part A: Checking Paramecium PhenotypesThe TA will describe the expected phenotypes of the Test and Control cells. View the cells in a depression slide under a dissecting microscope to confirm the expected phenotypes of both treatments.

1. Take 500 µL of Test and Control cultures and transfer to a depression slide. Mark the depression slides as Test and Control.

2. Transfer cells to 500 µL of Resting Solution in another depression slide. Try not to transfer much culture medium.

3. Wait 10 minutes.4. In the meantime, take 500 µL of 1mM BaCl2 solution to another depression slide.

Label the depression slide Test and Control.5. After 10 minutes, transfer 1 cell at a time from the resting solution to the BaCl2

solution. Note the swimming behavior of each. First do this with control cells, then with test cells.

6. Check at least 15 cells. – (Each person do 10 Control, 10 Test. 20 Total #’s.)

Part B: Filtering and Washing the Paramecium (Your TA will demonstrate procedure in MLS 224)

1. Filter cells through a folded square of small Kimwipe into a 100 mL pear-shaped centrifuge tube

2. Balance centrifuge tubes and centrifuge at ¾ speed for 2 minutes in the IEC HN-SII centrifuge. Balance with LB.

3. Using a glass Pasteur pipettes remove the cells collected at the bottom of the tube. (Your TA will demonstrate how to use circular motion to aid in removing concentrated amount of cells). Dump the supernatant into the waste container provided and then place the pipette containing the cells back into the empty pear-shaped centrifuge tube.

4. Add approximately 100 mL Dryl’s solution to the empty pear-shaped centrifuge tube and pipette in the cells to mix.

5. Balance and centrifuge the tubes at ¾ speed for 2 minutes in the IEC HN-SII centrifuge.

6. Pipette out cells using the demonstrated technique and transfer to a 1.5 mL microcentrifuge tube. Continue to next section and start RNA Prep within a few minutes of collecting cells.

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Part C: RNA Prep (To be completed in MLS 007A by students) 1. Disinfect lab bench, pipettes, Sharpie, and gloves of all RNases. Spray surfaces

with “RNase Away” and then wipe with paper towel. It is crucial that the following protocol be performed in an RNase-free environment. Your TA will explain the importance of RNase-free conditions.

2. Centrifuge cells for 10 seconds and remove the supernatant using a pipette. Transfer approximately 100 µL of the tightly packed cells to a 0.5 mL centrifuge tube.

3. Add 350 L of the lysis buffer (RA1) to the 0.5 mL tube of cells4. Next, add 3.5 L of β-Mercaptoethanol. Use β-Mercaptoethanol in the hood

and place all pipette tips that come into contact with β-Mercaptoethanol into designated waste container!

5. Use a 1 mL syringe to mix the solution approximately 3 times. 6. Place a pink filter column inside a collecting tube and pipette the cell solution

onto the center of the filter. Label both the collecting tube and the filter. 7. Centrifuge for 1 minute at 12,000 rpm8. Remove filter and discard9. Add 350 L of 70% ethanol to the sample in the collecting tube. Pipette up and

down to mix10. Set up a blue spin column with collecting tube and label both. 11. Transfer the sample to the blue spin column and centrifuge for 1 minute at 8,000

rpm12. Place column into a new collecting tube and label. Discard supernatant and used

collecting tube. 13. Add 350 L Membrane Desalting Buffer (MDB) and centrifuge at 12,000 rpm for

1 minute14. Add 90 L of DNase Reaction Buffer and 10 L of DNase I stock solution to a

fresh 0.5 mL microcentrifuge tube.15. Take 95 L of the DNase Reaction Mixture and add it to the center of the blue

spin column. Do not contact column and make sure DNase is absorbed. Let sit at room temperature for 45 minutes.

16. Add 200 L RA2 Buffer to the spin column and centrifuge for 1 minute at 8,000 rpm. (If the tip of the column outlet comes into contact with the flow-through for any reason, discard the flow-through and centrifuge again at 12,000 rpm for 1 minute.)

17. Place spin column into a new collecting tube18. Add 600 L of RA3 Buffer to spin column and centrifuge for 1 minute at 8,000

rpm19. Discard the flow-through and place the column back into the collecting tube. 20. Add 250 L RA3 Buffer to the spin column and centrifuge for 2 minutes at 12,000

rpm.21. Place the spin column into the supplied RNase-free 1.5 mL microcentrifuge tube

and label.

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22. Elute the RNA by adding 20 L of RNase-free water directly into the center of the spin column. Centrifuge for 1 minute at 12,000 rpm

23. Take the liquid collected in the collecting tube and transfer to the top of column to elute again. Wait 1 minute and then centrifuge at 12,000 rpm for 1 minute.

24. Discard spin column and aliquot 2 µL to a 0.5 mL tube for the lab tech to nanodrop.

25. Place the tube in the -80C freezer.

2.10 Making cDNA __________ Your TA will determine the total RNA yield for each of your samples using the Nanodrop. The concentration will be reported in ng/L.

1. Calculate the volume of RNA needed to equal 5 ug per tube using the RNA concentration provided by the TA.

2. Disinfect lab bench, pipettes, Sharpie, and gloves of all RNases. Spray surfaces with “RNase Away” and then wipe with paper towel. It is crucial that the following protocol be performed in an RNase-free environment.

3. Set up the RT-PCR using 0.5 mL tubes and the following table. Tube 1 will contain test RNA and Tube 2 will contain control RNA. Tube 3 is the negative control which will contain test RNA and no SuperScript III.

*Your TA will inform you on how much RNA to add to each tube based on the results from the Nanodrop. Calculate the volume of water that you need based on the amount of RNA added. Add this chart to your lab notebooks with calculated volumes.

4. Incubate at 65C for 5 minutes then put on ice and add:

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Reagents (µL) Tube 1 (Test) Tube 2 (+ Control) Tube 3 (- Control)RNA * 5 µgWater *50 µM Oligo dT 1 1 110 mM dNTPs 1 1 1Total volume 13 13 13

5. Place tubes in thermocycler and set to the following conditions: cDNA synthesis Conditions

25C 5 min50C 30 min55C 30 min70C 15 min

6. Remove tubes from thermocycler and place at -20C.

2.11 Template Control and Endogenous PCR _____ Part A: Serial Dilutions

1. Make serial dilutions of both the Test and +Control cDNA according to the following chart:

Serial Dilution cDNA H2O1:10 2 µL of cDNA 18 µL1:50 2 µL of previous dilution 8 µL1:100 4 µL of previous dilution 4 µL1:500 2 µL of previous dilution 8 µL

2. Keep tubes on ice while preparing PCR tubes

Part B: Template Control PCR

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Reagents (µL) Tube 1 (Test) Tube 2 (+ Control) Tube 3 (- Control)5X First-Strand Buffer 4 4 40.1 M DTT 1 1 1RNase Out 1 1 1SuperScript III 1 1 0Water 0 0 1Total Volume 20 20 20

Calmodulin is a protein highly expressed in paramecium. We will detect the mRNA levels of the calmodulin gene in both the Test and +Control to determine if there are equal concentrations of mRNA in both the Test and the +Control.

Calmodulin primers: Forward: 5’ CTG AAG CTG AAC TTC AAG 3’Reverse: 5’ TCA TTT AGA AAC CAT CAT TCT 3’Product length: 330-350 bp

1. Set up and label 9, 0.5 mL tubes according to the table below.

Calmodulin PCR (Note the endogenous set up is different)Tube Treatment µL of cDNA µL of Master Mix1 1X Test 1 492 1:10 Test 1 493 1:100 Test 1 494 1:500 Test 1 495 1X +Control 1 496 1:10 +Control 1 497 1:100 +Control 1 498 1:500 +Control 1 499 1X -Control 1 49

2. Each tube will contain 1 µL of the respective cDNA dilution and 49 µL of master mix. The master mix contains the reagents necessary for the PCR. Make the master mix in a 1.5 mL tube by adding:

Sterile dH2O 375 µL10X PCR buffer 50 µL25mM MgCl2 30 µL10 mM dNTPs 10µLCalmodulin F primer 10 µLCalmodulin R primer 10 µLTaq Polymerase 5 µL

3. Mix the solution well by pipetteing up and down.4. Add 49 µL of master mix to each labeled tube. 5. Add 1 µL of respective cDNA dilution to each tube. Mix well.6. Spin tubes in microcentrifuge for 10 seconds.7. Place tubes in PCR machine making sure the caps are completely closed and the

contents of the tube are at the bottom of each tube and not stuck up on the sides or in the cap.

8. Run PCR with the following conditions:Calmodulin PCR Conditions

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95C 5 min Initial Denature95C 1 min

24X42C 1 min72C 40 sec72C 10 min Final Elongation4C HOLD ****************

Part C: Endogenous gene PCR

The endogenous PCR will allow us to check the mRNA levels and see if the gene was down regulated due to RNA interference.

1. Set up and label 9, 0.5 mL tubes according to the table below.

Endogenous PCRTube Treatment µL of cDNA µL of Master Mix1 1X Test 0.5 24.52 1:10 Test 0.5 24.53 1:50 Test 0.5 24.54 1:100 Test 0.5 24.55 1X +Control 0.5 24.56 1:10 +Control 0.5 24.57 1:50 +Control 0.5 24.58 1:100 +Control 0.5 24.59 1X -Control 0.5 24.5

2. Each tube will contain 0.5 µL of the respective cDNA dilution and 24.5 µl of master mix. The master mix contains the reagents necessary for the PCR. Make the master mix in a 1.5 mL tube by adding:

Sterile dH2O 172.5 µL (group dependent)10X PCR buffer 25 µL25mM MgCl2 20 µL (group dependent) 10 mM dNTPs 5 µLEndogenous F primer 5 µLEndogenous R primer 15 µLTaq Polymerase 2.5 µL

3. Mix the solution well by pipetteing up and down.4. Add 24.5 µL of master mix to each labeled tube. 5. Add 0.5 µL of respective cDNA dilution to each tube. Mix well.6. Place tubes in PCR machine making sure the caps are completely closed and the

contents of the tube are at the bottom of each tube and not stuck up on the sides or in the cap.

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7. Run PCR with the conditions you determined during 2.4:

Endogenous PCR Conditions Temp Time (min) Cycle

Initial Denature

30X

Final ElongationHOLD ****************

8. When the PCR is complete your TA will remove the PCR product tubes from the thermocycler and place at 4C.

2.12 Template Control Gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

1. Make 300 mL 1X TAE.2. Prepare a 2% agarose gel with a 12-well comb. Note: The 2% agarose solution

will solidify quickly! Pour gel while still relatively hot. 3. Remove 10 µL of PCR product from each tube; add to 2 µL of sample buffer.

Store the remaining PCR product at 4ºC.4. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be loaded directly

on the gel. 5. Once your gel is set, remove the comb and place the gel in the running box. 6. Cover the gel with 1X TAE buffer and load your samples.7. Run gel at 120 volts for 45 minutes to 1 hour.8. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium

bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.9. Destain, examine, and photograph your gel.

2.13 Endogenous mRNA Gel HAZARDOUS CHEMICAL INFO:-Ethidium Bromide is an extremely toxic carcinogen. WEAR GLOVES when handling, and dispose of everything that has contacted EtBr in the appropriate solid waste container.

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1. Make 300 mL 1X TAE.2. Prepare a 1.5% agarose gel with a 12-well comb. Note: The 1.5% agarose

solution will solidify quickly! Pour gel while still relatively hot. 3. Remove 10 µL of PCR product from each tube; add to 2 µL of sample buffer.

Store the remaining PCR product at 4°C.4. Obtain an aliquot of 100bp ladder from the Lab Tech; this will be loaded directly

on the gel. 5. Once your gel is set, remove the comb and place the gel in the running box. 6. Cover the gel with 1X TAE buffer and load your samples.7. Run gel at 120 volts for 45 minutes to 1 hour.8. Once the electrophoresis is complete, stain your gel for ~15 minutes in ethidium

bromide. WEAR GLOVES! Ethidium bromide is a mutagen and carcinogen.9. Destain, examine, and photograph your gel.

2.14 Analyze Data_____________________________________________Work with your partner and discuss the results. Your TA will be there to answer any questions you may have.

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Module 3Proteomics

Introduction In this module, you will compare the proteins found in wild type and mutant Paramecia cilia. You will run a one dimensional polyacrylamide gel, cut out the bands of interest, and compare the proteins present in those bands using mass spectrometry.

3.0 Preparation of Cilia (Done for you) _____ Your TA will prepare the cilia from Paramecium using the following protocol:

1. Filter cell culture by slowly pouring paramecium cell culture through a funnel lined with cheese cloth sandwiched between 2 large kimwipes into a clean 2800 mL flask.

2. Centrifuge cell culture to concentrate the cells. 3. Pipette cells into a beaker containing 100 mL of room temperature Dryl’s

solution. Mix the solution and then pour through a Kimwipe in a funnel into a clean pear-shaped centrifuge tube . Centrifuge for 2 minutes at ¾ speed. Remove trichocysts (fluffy layer on top of pellet) and put in waste container. Transfer cells to clean Dryl’s buffer. Mix and centrifuge again. Do for a total of 3 washes.

4. Add cells to a flask containing 40 mL cold Dryl’s solution, then add 40 mL cold STEN buffer. Keep on ice for 10 minutes. The cells should be immobilized within 10 minutes.

5. Add 16 mL Cilia Shock Buffer to cells, swirl the beaker and place on ice for 2-5 minutes. During this time the cells will deciliate – examine under inverted microscope to watch the progress of deciliation, and to make sure that the cell lysis is minimal (preferable <1%). If cells begin to lyse, spin down immediately.

6. Transfer cell solution to pear-shaped flasks and centrifuge for 2 minutes on full speed. Pour off supernatant into clean pear-shaped flask, leaving cell bodies in old flask. Spin supernatant again for 2 minutes at full speed.

7. Transfer supernatant to 30 mL Cortex tubes with rubber sleeves. Spin in Beckman J2-21 Centrifuge (JA-17 rotor) at 14,500 rpm for 20 minutes.

8. Add 3 mL of 10mM Tris EDTA pH 8.3 to one tube and vortex for 5 minutes. Transfer the resuspension to the next tube and vortex for 5 minutes. Continue to consolidate the pellets until all pellets have been resuspened.

9. Rinse the empty tubes with 2 ml Tris-EDTA pH 8.3 to collect any residual cilia. The total volume of the sample should now be 5ml.

10. Place sample in a fresh 15ml Corex tube and wash the last tube with 1-2 ml Tris EDTA pH 8.3.

11. Spin at 19,500 rpm in JA-20 rotor for 30 minutes.12. After the spin, pour off the supernatant and resuspend the pellet in 400 - 600 µL

of 10mM Tris (pH 8.0). Be sure the suspension is homogenous.

34

13. Store at -80°C for future use.

3.1 Ultracentrifugation_ ________________________________________ Each group will be provided with a cilia protein sample. Three groups will be given wild type samples and three groups will be given Pawn A samples. Keep these on ice.

1. You are provided 66% sucrose. In three 15 mL tubes create three concentrations of sucrose (55%, 45% and 20%) using the calculations in Appendix J.

2. Create the sucrose gradient by first adding 0.7 mL of 66% sucrose to the Ultraclear Ultracentrifuge tube, then 1.4 mL 55% sucrose, then 1.4 mL 45% sucrose and finally 0.7 mL 20% sucrose.

3. Carefully place 200 to 300 µL of your sample on top of the sucrose gradient. Rinse the empty protein sample tube with 100ul of 10mM Tris (pH 8.0) and add to gradient.

4. Balance the ultracentrifuge tubes in their buckets with 10mM Tris (pH 8.0). Note the number on the bucket that corresponds to your sample.

5. YOUR TA WILL HELP YOU SET UP THE ULTRACENTRIFUGE ROTOR AND PLACE THE ROTOR IN THE CENTRIFUGE!!

6. Ultracentrifuge (Sw60Ti rotor) at 45,000 rpm at 4°C for 1.5 hours.7. After centrifugation there should be three different samples at the different

interfaces. See Appendix K. We are only interested in collecting the pure ciliary membrane which is between the 20% and 45% sucrose gradients.

8. Use an insulin syringe to poke a hole in the side of the ultracentrifuge tube and suck out the white, cloudy interface between the 20% and 45% sucrose gradients. Place the collected sample into a fresh 15ml corex tube.

9. Add 12 mL of 10mM Tris (pH 8.0) to each tube and invert to mix well. 10. Balance the tubes in their rubber sleeves. 11. Centrifuge in the Beckman J2-21 (JA- 17 rotor) at 17,000 rpm at 4°C for 30

minutes.12. Immediately remove the samples, pour off the supernatant and discard in the

waste container. Allow tubes to rest upside down on a kimwipe for a minute and then place on ice.

13. Resuspend the pellet in 100 µL of Membrane Buffer with protease inhibitors. 14. Remove 15 µL and place in a labeled microfuge tube for the Pierce Protein Assay.

Store the tube at -80°C.15. Store remaining samples at -80°C.

35

3.2 Preparation of Resolving Gel HAZARDOUS CHEMICAL INFO:-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container.

1. Obtain one 1.5mm spacer glass plate and one short glass plate; wipe with methanol and a Kimwipe until you hear a “squeaky” noise. Handle glass plates at edges. Wear gloves!

2. Assemble the gel casting apparatus (See diagram in Appendix L). Assemble on a flat surface and then clamp shut.

3. Insert the 1.5mm comb and use a Sharpie to draw a line across the glass 0.7 cm below the comb. Once the line is drawn, remove the comb.

4. Test to see if the apparatus is leak-proof. Squirt some water in between the glass plates and look for leaks. If leaks occur, a tighter seal must be achieved. Make sure to remove the water before pouring the gel. Use a Kimwipe to remove water droplets from between the plates of glass.

Before you pour your gel, have the TA or lab tech check your apparatus!

5. Prepare a 12% resolving gel in a 15 mL tube according to the following directions. DO NOT ADD THE FRESHLY MADE AMMONIUM PERSULFATE UNTIL YOU ARE READY TO POUR THE GEL

Sterile dH2O 3.29 mL 4X Resolving Buffer pH 8.9 2.60 mL

30% Acrylamide stock 4.00 mL TEMED 10 L

Last: Fresh 10% Ammonium persulfate 100 L

6. Gently swirl the solutions to mix WELL. 7. Using a Pasteur pipette, pour the gel by allowing the acrylamide solution to

run down along the side of the spacer. Add the acrylamide solution until it is just barely above your Sharpie line. Try to avoid making bubbles.

8. Overlay the acrylamide with dH2O. Do this by gently adding the dH2O with a glass Pasteur pipette. You will be able to see a distinct line between the dH2O and the resolving gel solution.

9. Allow the gel to polymerize for AT LEAST 30 minutes (Any extra acrylamide mix in your tube will be a good gauge for polymerization. Make sure the cap is on).

36

10. Once your gel has polymerized, wrap the gel/casting apparatus in a damp paper towel and then plastic wrap. Label appropriately. Store in the cold room.

3.3 Pierce Protein Assay Pierce Protein Assay (See directions in Appendix M)Use the chart provided to develop a standard curve using BSA standards and to determine protein concentration.

1. Dilute protein sample: Make 10X and 20X dilutions for each protein sample. For example, to make a 20X dilution, add 5 L of your sample to 95 L of sterile dH2O. For a 10X dilution add 10 L of your sample to 90 L of sterile dH2O.

2. Make dye solution: Use Solutions A and B from the Pierce Protein Assay Kit. They should be mixed 50:1…but make up only the amount you will need. Mix the dye in a 50 mL tube.

3. Add BSA and dH2O according to the directions in Appendix M.4. Add 2 mL of the dye to each one of your samples and standards.5. Incubate at 37°C for 30 minutes. 6. Get OD values for standards and samples: TA will assist in the operation of the

spec. 7. Place your standard into a clean cuvette.

To clean the cuvette, rinse with dH2O. Make sure to dry the outside of the cuvette with a Kimwipe. Handle the cuvette only on the frosted sides.

8. Read OD at 562 nm. 9. Repeat for each standard and sample, including duplicates.

If only using 1 cuvette, make sure to rinse with dH2O between each standard.10. Once the standards are complete, read your samples. You should blank the

instrument with dH2O and dye. If using only 1 cuvette, make sure to rinse with dH2O between each sample.

11. Find the average of each standard and sample duplicates.12. Establish a standard curve using the OD values obtained with your BSA

standards: graph Concentration (x-axis) vs. OD (y-axis) on graph paper. Using this graph, calculate the protein concentrations in your three samples.

13. Use Excel to plot your data on a second chart (This is homework). Make sure to paste your Excel chart in your notebook properly labeled.

3.4 SDS-PAGE HAZARDOUS CHEMICAL INFO:

37

-Acrylamide is a neurotoxin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the acrylamide) in the appropriate waste container.-TEMED is highly toxic. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the TEMED) in the appropriate waste container. Do not inhale fumes.-Ammonium Persulfate is highly toxic upon contact with skin. WEAR GLOVES when handling, and dispose of all solid waste (pipets, gloves, or anything else that has come in contact with the APS) in the appropriate waste container. -Coomassie Blue Stain and Coomassie Blue Destain are highly flammable and irritating to the skin. WEAR GLOVES when handling and dispose of in appropriate waste container.

1. Remove the resolving gel from the cold room and pour a 4% stacking gel. 2. Mix the following components in a 15 mL tube. 3. Sterile dH2O 6.10 mL

4X Stacking Buffer pH 6.8 2.50 mL

30% Acrylamide stock 1.30 mL TEMED 10 L

Last: Fresh 10% Ammonium persulfate 50 L

4. Before adding the ammonium persulfate, pour the dH2O off the resolving gel and dry with a Kimwipe.

5. Add the ammonium persulfate to your tube. Mix gently.6. Pour the stacking gel as you did the resolving gel all the way to the top of the

small glass plate. If it overflows when inserting the comb this is okay. Clean the comb thoroughly with ethanol before inserting.

7. Being careful to avoid making air bubbles, insert the clean comb until there is no air between the wells. This is VERY IMPORTANT; the stacking gel will not polymerize if the comb is not clean, or if there is air between the wells. Ask your lab tech to double check your set up.

8. Allow 30 minutes for the gel to polymerize. Thaw your protein samples on ice while waiting.

9. Once the gels are set, remove them from the casting stand and assemble in the gel box (See Appendix N). Note: Do not remove the comb yet.

10. Add 1X PAGE Running buffer to the upper chamber. The buffer level should be half way between the top of the big and small glass plates.

11. Add 1X PAGE Running buffer to the lower chamber until the appropriate level for the number of gels in the box is reached.

12. Carefully remove the comb.

38

Gel Set UpYour TA will help you determine how much sample you are going to load based on the concentration of your protein sample. Each gel will have test and control sample lanes and a protein marker. Your TA will help you to equalize the concentrations of test and control so that the lanes on the gel are comparable . Any empty lanes should be filled with sample buffer.

1. Calculate the volumes of sample, 10X SDS sample buffer, and water needed for each tube:

-Each tube needs 60 µg of protein total. Using the concentrations you calculated off of your standard curve, calculate how many microliters equals 60 µg.

2. Add the calculated volumes of water, protein, and 10X SDS sample buffer to a 1.5 mL tube. Place the remaining protein samples in the freezer.

3. Add 1 µL of β-Mercaptoethanol to protein sample mixture.4. Boil samples for 5 minutes right before you are ready to load. After boiling, keep

samples on ice while loading gel. 5. Using gel loading tips, load 20 μL of each of your samples into the gel in the

following order: Lane 1, 3, 4, 6, 7, 9 and 10: SDS sample buffer Lane 2: Prestained protein markerLane 5: 60 µg WT Lane 8: 60 µg PwA

6. Run the gel at 50 mA for 1-1 ½ hours.7. Remove gel carefully from the gel apparatus. Use a razor blade to cut the

stacking gel portion away. Dispose of the stacking gel in the appropriate waste container.

8. Put the gel into a plastic container and cover with Coomassie stain. Stain overnight.

9. Pour off stain into original container.10. Add destain to gel. Wash and discard destain in correct waste container. 11. Rinse gel with destain again and decant destain into correct waste container. 12. Submerge the gel in more destain. Rock gently overnight. 13. The Lab tech will save these gels in destain until the next lab.

39

3.5 Cutting out gel bands and trypsinizing proteins in preparation_ for Mass Spectrometry_________________________________________HAZARDOUS CHEMICAL INFO:-Acetonitrile is poisonous. It also tends to leak from pipet tips when being measured. Always wear eye protection and gloves when handling it or transferring it. Always dispose of acetonitrile in its special waste receptacle. If you get acetonitrile on your gloves, change your gloves.

1. Clean a glass plate with soap and water and rinse thoroughly with sterile water. Rinse again with 95% ethanol. Let the plate dry.

2. Carefully remove the gel from its container and place on the glass plate. 3. Label a 1.5 mL microcentrifuge tube with appropriate identification for each

protein band to be analyzed. Select the razor blade and clean with 95% ethanol before using and between cutting each band.

4. Select bands of interest based on visible differences in intensity of Coomassie staining. Cut out the selected gel bands into small pieces (1mm) and transfer them to the labeled tubes carefully.

5. Add 900 µL of HPLC-grade water to each tube. Incubate at room temperature for five minutes.

6. Centrifuge at high speed for 30 seconds. Using your pipettor set to 1000 µL, carefully remove the water and discard it in the waste container provided. Use a new pipet tip for each sample. Be careful not to lose the gel in this process.

Note: The following steps use acetonitrile, which is poisonous. It also tends to leak from pipet tips when being measured. Always wear eye protection and gloves when handling it or transferring it. Always dispose of acetonitrile in its special waste receptacle. If you get acetonitrile on your gloves, change your gloves.

7. Add 750 µL destain solution (50mM ammonium bicarbonate, 50% acetonitrile) to each tube. Close the tube cap and mix gently by inversion. Incubate the tubes at 37°C for 20 minutes.

8. At the end of 20 minutes inspect the gel. If it is still very blue, repeat the destain process with more destain reagent. To do this, briefly centrifuge the tube, carefully pipet off the liquid, add 750 µL new destain reagent and incubate for an additional 15 minutes. Make sure you put the discarded destain solution in the provided waste receptacle. Repeat one more time if needed. Change gloves if you get acetonitrile on them and wash skin with water if it contacts the acetonitrile.

9. When the gel sample is clear and no longer contains blue color, centrifuge the tube at high speed for 30 seconds. Carefully remove all the destain solution and discard it in the waste receptacle.

10. Add 100 µL of 100% acetonitrile to each tube. (The gel pieces should be entirely immersed, if not add more). The gel pieces will turn white as they dehydrate. Incubate the tubes for 2 minutes at room temperature.

40

11. Centrifuge for 30 seconds and carefully pipet off all the acetonitrile. Discard in the provided waste receptacle. Use a 200 µL pipettor to carefully remove the residual liquid from the tube.

12. Open lids and place tubes in Speed Vac for 5 minutes to dry samples completely. Close the lids before proceeding to the next step.

13. Place the tubes on ice for 5 minutes. Add 25 µL ice cold trypsin/50 mM ammonium bicarbonate solution to each tube. Incubate on ice for five minutes. Add 25 µL cold 50 mM ammonium bicarbonate solution to each tube. Make sure the gel is completely immersed in the solution. Add more ammonium bicarbonate solution if necessary.

14. Incubate the tubes on ice for an additional 30 minutes.15. Transfer the tubes to a 37°C incubator. Incubate the tubes overnight (8-16 hrs).

The Next Day:16. Spin the tubes of gel slices for 1 minute at full speed. Transfer all of the peptide

solution (supernatant) to a properly labeled 0.5 mL tube. (If it is too difficult to remove the liquid because the gel pieces are too big, wait until the next step to pull off the peptide solution.)

17. Add 150 µL of 50% Acetonitrile / 2.5% formic acid to the tubes of gel slices and vortex.

18. Spin these tubes for 5 minutes at full speed and transfer the peptide solution (supernatant) to their respective 0.5 mL sample tubes.

19. Add 100 µL of 100% Acetonitrile to the tubes of gel slices and vortex.20. Spin for 5 minutes at full speed and transfer the peptide solution (supernatant)

to the respective 0.5 mL sample tubes. Save the tubes of gel pieces at room temperature for later extraction if needed.

21. Dry the samples using the Speed Vac for 2 to 4 hours.22. Your TA will bring your dried samples to the UVM Proteomics Core Facility for

processing.

3.6 Mass Spec Tour the UVM Proteomics Core Facility in MLS 337 and meet to discuss how mass spec works.

3.7 Mass Spec Results Discussion_______________________________Experienced proteomics personnel will discuss how to analyze the results you receive from the Mass Spec.

3.8 Analyze results Your TA and professor(s) will help you to analyze your results from mass spec, and determine which proteins are differentially expressed.

41

Appendix A: Solutions Guide

GENERAL SOLUTIONS

Ampicillin Stock100 mg/ml stock solution: for example, 0.5g ampicillin sodium salt into 5 ml dH2O. Filter sterilize, and store at -20°C.

LB amp100 µg/ml final concentration: 1:1,000 dilution of ampicillin stock into LB broth.

When making LB amp plates, add 1 ml ampicillin stock (100mg/ml) into 1L LB Agar broth. *NOTE: Ampicillin is heat-sensitive, so LB agar broth must be cooled to 60°C after coming out of the autoclave, before the ampicillin is added. Setting the water bath to 60°C and letting the LB agar broth cool in there for an hour is a good way to ensure the LB agar doesn’t solidify.

When making LB amp broth, add 1ul of ampicillin stock (100mg/ml) for each 1ml of LB broth.

TE bufferNeed (final conc.): 10mM Tris-Cl (pH 7.5), 1mM EDTA (pH 8.0)Make from liquid stocks of Tris-Cl and EDTA5ml 2M Tris-Cl (pH 7.5)2ml 0.5M EDTA (pH 8)993ml dH2O

2M Tris-Cl (pH 8.0)177.6g Tris-Cl10.6g Tris-baseIn ~950mL sterile dH2O**pH 8.0**Bring up to 1L with sterile dH2O

0.5M EDTA (pH 8)18.6g EDTA disodium salt (FW= 372.2)In ~75ml sterile dH2OHeat in microwave to dissolve salt***bring pH to 8.0***Bring up to 100mL with sterile dH2O

42

50X TAE stock (pH 8.5)242g Tris Base (FW= 121.14)In ~700ml sterile dH2OCarefully add 57.1mL Glacial Acetic Acid100mL 0.5M EDTA (pH 8.0)Bring up to 1L with sterile dH2O

pH 8.5, but no adjustment needed*Dilute 50X TAE stock 1:10 for a 5X stock*

6X DNA sample buffer0.25 g Bromophenol Blue40 g Sucrose100 ml dH2O

λ Hind III markerWant a final concentration of 100 ng/µLMake: 96 µL λ Hind III marker (Invitrogen stock)

320 µL TE68 µL 6X DNA sample buffer

20X SSC (pH 7)175.3 g NaCl88.25 g Na3 Citrate•2H2O1 L dH2O**pH 7.0**

2X SSC10ml 20X SSC90ml dH2O

10X TBS (pH 7.4)

25.1g Tris HCl4.8 g Tris Base80 g NaCl 800mls of H2O.Make volume up to 1 L with high purity distilled or deionized water.

Once prepared, TBS is stable at 4°C for 3 months.

43

10X TBS-T10X TBS, 1% Tween-20

10 ml Tween-20 (use large orifice tips to pick up Tween)1L 10X TBS

Phosphate Buffer (PBS)4.0g NaCl0.1g KCl0.72g Na2HPO4

0.12g KH2PO4

In ~400ml sterile dH2O**pH 7.4**Bring to 500ml with sterile dH2O

MODULE 1

1.1: Isolation of plasmid DNA

Solution I0.50 g D-Glucose0.625 ml 2M Tris-Cl (pH 8)1 ml 0.5M EDTAAdd dH2O to make total volume 50ml**add 5 mg/ml lysozyme just before use**

Solution II2 ml 1M NaOH1 ml 10% SDS7 ml dH2O**Prep fresh**

5M Potassium Acetate 29.5 ml glacial Acetic Acid 100 ml dH2O Add KOH pellets until pH=4.8Store in refrigerator

44

Heat-treated RNase A (100mg/ml)Dissolve 100 mg (0.1g) of pancreatic RNase A in 1 ml 10mM Tris-Cl/15mM NaCl. Store at -20 °C. Before use, heat RNase A in 100°C heat block for 15 minutes and allow tubes to cool slowly to RT.

10mM Tris-Cl/15mM NaCl0.1576 g Tris-Cl0.0876 g NaCl100ml dH2O

Salt Saturated PhenolTris buffered Phenol pH 6.6/7.98-Hydroxyquionoline added until dark yellow/orange color

Chloroform:Isoamyl Alcohol (24:1)480 ml Chloroform20 ml Isoamyl Alcohol

TE bufferSee general solutions section

1.4: Transformation

50 mM CaCl2

0.73 g CaCl2

100 ml dH2O** Make fresh**

Transformation buffer1ml 100 mM CaCl2

1 ml 100 mM Tris (pH 8.0)1 ml 100 mM NaCl7 ml dH2O**Store at 4°C**

45

1.6: Secondary selection of transformed bacteria

0.145 M Sterile Saline (pH ~7)4.25 g NaCl500 ml dH2O** Autoclave to sterilize**

1.8: Cracking gel

Cracking Buffer (pH 6.8) 0.788 g Tris-Cl1.0 g SDS0.058 g Na2EDTA•2H2O13.6 g Sucrose0.1 g Bromophenol Blue100 ml dH2O** pH 6.8**

1.9: Biotin labeling of DNA

3 M Sodium Acetate (pH 4.8)24.6 g Sodium Acetate100 ml dH2O** pH 4.8**

1.11: Southern blot

Cracking BufferSee 1.8 above

0.5M NaOH/0.8M NaCl20 g NaOH46.752 g NaCl1 L dH2O

0.5M Tris/1.5M NaCl (pH 7)250 ml 2 M Tris-base solution (pH 8)87.6 g NaCl750 ml dH2O** pH 7**

46

10X SSCSee general solutions for 20X SSC

1.13: Hybridization of Southern Blot

Prehybridization Solution (per group, prep fresh)5 ml Formamide2.5 ml 20X SSC0.5 ml 100X Denhardt’s solution (doesn’t keep more than 24 hours!)0.25 ml 1M Phosphate Buffer200 µL Herring sperm DNA (2mg/ml, made fresh), freshly denatured

Hybridization Solution (per group, prep fresh)4.5 ml Formamide 2.5 ml 20X SSC0.1 ml 100X Denhardt’s solution (doesn’t keep more than 24 hours!)0.4 ml 1M Phosphate Buffer1.5 ml dH2O200 µL Herring sperm DNA (2mg/ml), freshly denaturedBiotin-labeled probe DNA

100X Denhardt’s Solution0.2 g Ficoll 0.2 g Polyvinylpyrrolidone0.2 g Bovine Serum Albumin (BSA)10ml sterile dH2O**Doesn’t keep more than 24 hours**

1.14: Detection of DNA

2X SSC/0.1% (w/v) SDS 50 ml 20X SSC450 ml dH2O0.5 g SDS

0.2X SSC/0.1% (w/v) SDS 5 ml 20X SSC495 ml dH2O0.5 g SDS

0.16X SSC/0.1% (w/v) SDS 4 ml 20X SSC496 ml dH2O0.5 g SDS

47

1.15: Development of Blot

Buffer 1 : Final Concentration: 0.1 M Tris-Cl 0.15 M NaCl

8.7 g NaCl15.764 g Tris-Cl1 L dH2O

Buffer 2: 3% (w/v) BSA in Buffer 13g BSA per 100 ml Buffer 1**Doesn’t keep more than 24 hours, prep fresh**

SA-AP: **Needs to be made immediately before use**1 µL SA-AP per 1 ml Buffer 2 (approx. 10 ml needed per group)

Buffer 3Final concentration: 0.1M Tris-Cl pH=9.5

0.1M NaCl 50 mM MgCl2

15.764 g Tris-Cl5.844 g NaCl10.15 g MgCl2

1 L dH2O

1.17: PCR

20 µM Primer Dilutions (from 500 µM stock primers)5 µL 500µM stock120µL dH2O**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller batches**

10 mM dNTP mix (from 100 mM individual dNTP stocks)500µL dCTP500µL dTTP500µL dATP500µL dGTP3 ml dH2O *1 µL of 10mM dNTP mix per 50µL reaction*

48

MODULE 22.2 PCR

20 µM Primer Dilutions (from 500 µM stock primers)5 µL 500µM stock120 µL dH2O**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller batches**

10 mM dNTP mix (from 100 mM individual dNTP stocks)500 µL dCTP500 µL dTTP500 µL dATP500 µL dGTP3 mL dH2O*1 µL of 10mM dNTP mix per 50 µL reaction*

2.7 Induce Bacteria and Feed to Paramecium

0.2 M IPTG750 µL dH2O37.5mg IPTG

Wheat cultureWheat grass tea buffered with:3.75mM Na2HPO4+7H2O3 mg/L Stigmasterol

Dryl’s solution1mM Na2HPO4

1mM NaH2PO4

1.5mM CaCl2

2mM Sodium CitratepH 6.8

Stigmasterol5 mg/mL dissolved in 100% ethanol

49

2.9 Harvest cells and Isolate RNA

Resting Solution (5.1.1) 1mM Citric acid1mM Calcium hydroxide1mM Tris base5mM KCl pH 7.04

5mM BaCl21mM Citric acid1mM Calcium hydroxide1mM Tris base5mM BaCl2

pH 7.09

2.10 Making cDNA

50uM Oligo dT20µM Primer 5’ CGGCTCGAGTTTTTTTTTTTTTTTTTTTT 3’

10 mM dNTP mix (from 100 mM individual dNTP stocks)500 µL dCTP500 µL dTTP500 µL dATP500 µL dGTP3 mL dH2O

2.11 Template Control and Endogenous RT-PCR

20 µM Primer Dilutions (from 500 µM stock primers)5 µL 500µM stock120 µL dH2O**Primers should be stored long term at 500 µM conc. and diluted to 20 µM in smaller batches**

50

MODULE 3

3.0 Preparation of Cilia

Dryl’s solution1mM Na2HPO4

1mM Na2H2PO4

1.5mM CaCl2

2mM Sodium CitratepH 6.8

STEN Buffer17.12 g Sucrose0.316 g TrisCl0.068 g Na2EDTA0.036 g NaCl100 mL dH2O*pH to 7.5 and fill**store at 4C

Cilia Shock Buffer 180mM KCl 60mM CaCl2

Membrane Buffer0.331 g Tris-Cl0.048 g Tris base0.186 g KCl0.051 g MgCl2•6H2O0.019 g EGTA50 mL dH2O*pH to 7.4***add 1% Triton X-100 before use

LeupeptinDissolve in dH2O 1mg/mLStore at -20C in 1mL aliquots*Stable for 6 months*

51

Pepstatin A Dissolve in methanol 1 mg/mL(5mL methanol into 5 mg bottle and shake)Store at -20C in 1mL aliquots*Stable for 1 month*

100mM PMSF stock (Phenylmethylsulfonyl fluoride)0.871 g PMSF50 mL 100% ethanolAdd ~1.75 g of molecular sieves to each bottle to absorb any water*inactive in 30 minutes @ 4C in aqueous solution*

Preparation of Membrane Buffer with Protease Inhibitors1 mL Membrane Buffer2 µL Pepstatin2 µL Leupeptin10 µL PMSF2 µL IAA (0.02g/ml)*Add these immediately before use*

Tris EDTA0.0121 g 1mM Tris Base0.0034 g 0.1mM Na2EDTA100 mL dH2O*pH 8.3*

3.1 Ultracentrifugation

10mM Tris pH 8.05 mL 1M Tris, pH 8.0495 mL dH2O

66% Sucrose171 g Sucrose90 mL 10mM Tris pH 8.0Make the night before and place in shaker at 37o C overnight to dissolve

3.2 Preparation of Resolving Gel

52

4X Resolving Buffer (pH 8.9)18.17g Tris base10 g SDS (measure in hood)100 mL dH2O**pH 8.9**

3.4 SDS-PAGE

4X Stacking Buffer (pH 6.8) 6.055 g Tris base0.4 g SDS100 mL dH2O**pH to 6.8**

10X SDS Sample Buffer15.6 mL 8X TrisCl/SDS (pH 6.8)10 mL Glycerol2.0 g SDS5 mg Bromophenol blue*Add 1 µL of β-Mercaptoethanol to 10 µL of 10X SDS Sample Buffer*

8X TrisCl/SDS (pH 6.8)6.05 g Tris base dissolved in 35 mL dH2OAdjust the pH to 6.8 with concentrated HCl, and bring to 50 mL volume. 0.4 g SDS

5X Running Buffer7.55 g Tris Base36 g Glycine*Bring solution to 500 ml volume and stir. pH should be 8.3 but don’t adjust.*Add 2.5 g SDS. **Store at 4°C** Dilute to 1X before using

Coomassie Blue Stain200 mL Methanol50 mL Glacial Acetic Acid1 g Coomassie Blue250 mL dH2O

Coomassie Blue Destain200 mL Methanol

53

75 mL Glacial acetic Acid725 mL dH2O

3.5 Band Cutting and Trypsinization of Gel Plugs

Trypsin in 50mM ammonium bicarbonate Add the whole trypsin vial to 1.6 mL of 50mM Ammonium Bicarbonate*make up same day and keep on ice

Appendix B: Sterile TechniqueIt is very important in microbiology and genetics to work with pure cultures. Unfortunately, this is difficult. The world around us is covered with microorganisms. Microorganisms are even carried on dust particles in the air. In order to protect sterile broth, plates, slants and pure cultures from the microbes all around us, we must practice sterile (aseptic) technique. This simple means that sterile surfaces or sterile media must be protected from contamination by microbes in the air or residing on non-sterile surfaces. A simple example of the problem is that a sterile petri plate can become contaminated with bacteria when the lid is removed. In sterile technique, only sterile surfaces touch other sterile surfaces and exposure to the air is kept to a minimum.

In the classroom, you often need to practice sterile technique when you inoculate a pure culture of a microorganism into fresh medium. Sometimes this is a transfer to a tube of liquid broth and at other times, it is a transfer to a petri plate-containing agar. While there are other circumstances that require sterile technique, these are the most common and they will be described in more detail on the pages that follow.

Appendix C: Spread Plate Technique

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1. Dispense the appropriate volume of sample into the center of a sterile agar plate.

2. Dip the glass spreader (aka “hockey stick”) in alcohol. 3. Pass the spreader through the flame of a Bunsen burner to burn off the alcohol.

(This sterilizes the spreader).

***IMPORTANT***Keep the dish of alcohol behind the Bunsen burner.

Keep the alcohol dish covered when you are not using it.Keep your hand above the spreader at all times or flaming alcohol may roll toward

your hand.If the dish of alcohol catches on fire, cover the dish with the glass lid and it will go out.

4. Cool the spreader by touching it to the agar where there is no sample.5. Spread your sample over the entire surface of the agar.6. Sterilize the spreader before putting it back on the bench.

Appendix D: Use of a Rainin Pipettor

Take note: Never rotate the volume adjustor beyond the upper or lower range of the

pipette man, as stated by the manufacturer. Never use the pipette man without the tip in place; this could ruin the precision

piston that measures the volume of fluid. Never lay down the pipette man with filled tip; fluid could run back into the

piston. Never let plunger snap back after withdrawing or ejecting fluid; this could

damage the piston. Never immerse the barrel of the pipette man in fluid. Never flame pipette man tips.

If you drop your pipette man, the precision piston system can be damaged; therefore, if your pipette man is dropped, be sure to check the pipetting accuracy has not been affected.

Recommended Volume Ranges:

Model p10: 0.5-10 μL, the number after the decimal point is in red

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Model p20: 2-20 μL, the number after the decimal point is in redModel p200: 20-200 μL, there is no decimal pointModel p1000: 200-1000 μL, the numbers after the decimal point are in black

Pipetting Directions – Method1. Set the desired volume by holding the pipette man body in one hand and turning

the volume adjuster knob until the correct volume shows on the digital indicator. Approach the desired volume by dialing downward from a larger setting.

2. Press tip onto shaft by a slight twisting motion.3. Depress the plunger to FIRST POSITIVE STOP. This part of the stroke is the

calibrated volume displayed on the digital micrometer.4. Holding the pipette man vertically (never more than 20˚ from vertical), immerse

the tip just below the level of the liquid.5. Allow the pushbutton to return SLOWLY to the up position. Move the tip so that

it stays slightly below the level of the liquid as you draw up.6. Wait one to two seconds to ensure that the full volume of sample is drawn up

into the tip.7. Withdraw the tip from the sample liquid.8. To dispense the sample, place the tip end against the sidewall of the receiving

vessel and depress the plunger to the FIRST STOP. Wait one to two seconds. Then depress the plunger to the SECOND STOP, expelling any residual liquid in the tip.

9. With the plunger fully depressed, withdraw the pipette man from the vessel carefully with the tip sliding along the wall of the vessel.

10. Let the plunger return slowly to the UP position. If an air bubble is observed, re-pipette the sample.

11. Pre-rinsing the tip with the liquid being pipetted is recommended. A significant film may be retained on the inside wall of the tip, resulting in an error. Since the film remains relatively constant in successive pipettings with the same tip, refilling the tip a second time and using this quantity as the sample may obtain good reproducible results.

12. Discard the tip by depressing the tip ejector button smartly in the appropriate waste container.

Appendix E: Pipette ExercisesPipette Exercise #1

Determine and record the pipettor best suited for each of the measurements listed below.

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Add the indicated amounts to labeled microfuge tubes. Use the matrix below as a checklist while adding solutions to each microfuge tube.

Solution 1 Solution 2 Solution 3Tube A (green) 10 µL 25 µL 0.0963 mLTube B (red) 2 µL 0.015 mL 183 µLTube C (blue) 598.6 µL 0.200 mL 201.4 µL

Determine the total volume being added to each of the tubes. To check that your measurements are accurate, set a pipettor to the final volume and carefully withdraw the solution from each tube. Is the tip just filled? If measurements are inaccurate, repeat the exercise to obtain a near-perfect result.

Pipette Exercise #2

Using the p1000 and the p200 pipettors, perform the following:Set the pipettor to its maximum volume.Using water at room temperature, carefully pipette the water onto a weigh boat that you have tared (re-zeroed).Room temperature water has a density of approximately 1 gm/mL or 1 g/L. Therefore, you can determine the accuracy of your pipetting, e.g., 1000 L of water will weigh 1gm. Repeat the pipetting until you feel that you are reasonably accurate. Then record the weights of five successive pipettings. Determine the mean and standard deviation associated with your measurements.

Complete the following conversions:

1 L =_______mL 0.001 L=________mL10 L =_______mL 0.11 L =________mL100 L =_______mL 0.01 mL =________mL1000 L =_______mL 1 L =________mL

APPENDIX F: GST Plasmid Map

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APPENDIX G: GST Plasmid Map

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APPENDIX H: Frequently Used DNA/Protein Markers

Lambda DNA-Hind III Digest Prestained Protein Marker

100 bp DNA Ladder 1Kb DNA Ladder

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APPENDIX I: Streak Plate Method1 2

3 4

5

Flame loop in between each step except between 4 and 5.Do not flame loop between steps 4 and 5.

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APPENDIX J: PCR Reagents and Conditions for 1.17

Cycling Program: GST

94°C 5 min Initial Elongation94°C 1 min

5X50°C 1 min72°C 1 min

94°C 1 min25X51°C 1 min

72°C 1 min

72°C 10 min Final elongation4°C HOLD ***************

SAMPLESInitial Stock Concentration Components 1 2 3 4 5 6 7

Template DNA* 1 1 13

colonies3

colonies 1 1 10X Buffer 5 5 5 5 5 5 525mM MgCl2 0.5 1 3 3 3 3 310mM dNTPs 1 1 1 1 1 1 120µM Forward Primer 1 1 1 1 1 XXX 120µM Reverse Primer 1 1 1 1 1 1 XXX5U/µL Taq Polymerase 0.5 0.5 0.5 0.5 0.5 0.5 0.5 dH2O

*NOTE: Samples 1-3, 6 & 7: Use PLASMID DNASample 4: Transformed ColoniesSample 5: Non-Transformed ColoniesDESIRED FINAL VOLUME: 50 µL

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APPENDIX K: Sucrose Gradient Calculations

How to make __% Sucrose from 66% Sucrose

Add __ mL of 66% sucrose

Add __ mL 10mM Tris, pH 8.0

55% Sucrose 8.00 2.0045% Sucrose 6.35 3.6520% Sucrose 2.45 7.55

How to Create the Sucrose Gradient:

First add 0.7 mL 66% Sucrose,Then add 1.4 mL 55% Sucrose, Then add 1.4 mL 45% Sucrose,Finally add 0.7 mL 20% Sucrose.

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APPENDIX L: Sucrose Gradient Tubes after Ultracentrifugation

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APPENDIX M: Protein Gel Plate Setup

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APPENDIX N: PIERCE PROTEIN ASSAY for Module 3OD Value

Label L Alb (Stock 2mg/mL)

L dH2O

mL Dye

[Alb g/mL]

SeriesA

Series B

Average

S1 0 100 2 0S2 2.5 97.5 2 50S3 5.0 95.0 2 100S4 7.5 92.5 2 150S5 10.0 90.0 2 200S6 12.5 87.5 2 250S7 15.0 85.0 2 300

Label L Sample

L dH2O

mL Dye OD Values

Average OD Value g/mL in

Cuvette

g/mL Original Solution

2222

Label Average g/mL Original Solution of each treatment

g /LOriginal solution

L of protein that equates to 60 or 100 g total

WTPwA

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APPENDIX O: Protein Gel Running Setup

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APPENDIX P: RPM to G-Force Conversions

Equipment RPMG-Force (RCF)

Beckman J2-21 w/JA 14 rotor (250mL tubes) 5,000 3,840Beckman J2-21 w/JA 17 rotor (30 mL tubes) 5,000 3,440Beckman J2-21 w/JA 17 rotor (30 mL tubes) 9,500 12,400Beckman J2-21 w/JA 17 rotor (30 mL tubes) 15,000 31,000Beckman J2-21 w/JA-17 rotor (15 mL tubes) 17,000 39,000IEC Centra 7 Desktop w/ 15 mL tubes 2,800 1,098IEC Centra 7 Desktop w/ 50 mL tubes 2,800 1,098Eppendorf Centrifuge 5702 w/ 15 mL tubes 2,800 1,120Eppendorf Centrifuge 5702 w/ 50 mL tubes 2,800 1,180Eppendorf Centrifuge 5415 C w/ 1.5 mL tubes 14,000 15,980Sorvall Legend Centrifuge w/ 1.5ml tubes 14,000 18,800Ultracentrifuge 45,000 208,000Ultracentrifuge 21,600 45,000

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