Slide 1

WSSP-14 Chapter 1 Vectors and Libraries Slide 2 Today you will start doing something in the lab called " Molecular Cloning" Or " Genetic Engineering" Or " Recombinant DNA Technology " These techniques will allow you to study and manipulate individual genes Slide 3 For many years, biochemists had tried to purify genes. But they were frustrated because they are hard to purify. Slide 4 Because genes are composed of As, Cs, Gs, and Ts, they all pretty much are chemically alike. Also genes are parts of chromosomes. Chromosomes break easily and randomly, often in the middle of genes. So how did scientists eventually purify individual genes? Slide 5 Paul Berg Herb Boyer (Genetech) Stanley Cohen Genetic Engineering Nobel Prizes Slide 6 Amplify and Prep p. 2-1 Vector containing insert is transformed into E. coli for amplification and purification Slide 7 Vectors In order to study a DNA fragment (e.g., a gene), it needs to be amplified and eventually purified. These tasks are accomplished by cloning the DNA into a vector. A vector is generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli (can be a virus). p. 1-1 Slide 8 Plasmids Circular DNA molecules found in bacteria Replicated by the hosts machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication. Some plasmids are present in E. coli at 200-500 copies/cell p. 1-4 Slide 9 Plasmids also contain selectable markers. Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. Plasmid Engineering p. 1-4 Slide 10 Transforming plasmids into bacteria p. 1-2 Very inefficient: less than 1/1000 cells are transformed with the circular plasmid (linear does not transform) Slide 11 Normal Cell Cell treated with CaCl 2 Ca ++ Plasmid Transforming plasmids into bacteria Need to treat cells with Ca++ to transform plasmid (negatively charged membrane) (more postively charged membrane) Slide 12 Transforming plasmids into bacteria Very inefficient: less than 1/1000 cells are transformed with the plasmid How do you identify the few cells with the plasmid? Slide 13 Plasmids also contain selectable markers. Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. Plasmid Engineering p. 2-2 Slide 14 Plate cells on media with antibiotic Kills cells without the plasmid Slide 15 p. 1-2 Colony Cloning a DNA fragment Dead Cells Slide 16 Safety Features Modern cloning plasmids have been engineered so that they are incapable of transfer between bacterial cells Provide a level of biological containment. Naturally occurring plasmids with their associated drug resistance genes are responsible for the recent rise in antibiotic-resistant bacteria plaguing modern medicine. p. 1-3 Slide 17 X-gal LacZ -galactosidase, Jacob & Monod Slide 18 Screening for Inserts p. 1-3 Slide 19 Transform plasmid into bacteria Slide 20 DNA Libraries DNA library - a random collection of DNA fragments from an organism cloned into a vector Ideally contains at least one copy of every DNA sequence. Easily maintained in the laboratory Can be manipulated in various ways to facilitate the isolation of a DNA fragment of interest to a scientist. Numerous types of libraries exist for various organisms - Genomic and cDNA. p. 1-5 Slide 21 Construction and analysis of a genomic DNA library p. 1-5 Shotgun sequencing Slide 22 Construction and analysis of a genomic DNA library Slide 23 Construction and analysis of a genomic DNA library Slide 24 Construction and analysis of a genomic DNA library Slide 25 Want large clones to span the genomic DNA Slide 26 Sequencing the human genome cost $3 billion. Efforts are being made to cut the cost of sequencing a specific human genome to $1,000 (or less) Slide 27 We are not sequencing a genomic DNA library. We are sequencing a cDNA library What's that and what is the difference between the two? Slide 28 We are not sequencing a genomic DNA library. We are sequencing a cDNA library What's that and what is the difference between the two? A cDNA is a copy of RNA (usually mRNA) in the form of DNA. Slide 29 mRNA is a processed RNA transcript (in eukaryotes). It is intended to be translated into a protein. Slide 30 Construction of a cDNA library p. 1-6 Why are there blue colonies? Slide 31 Construction of a cDNA library Slide 32 Construction of a cDNA library ? Slide 33 Differences between a genomic and cDNA library p.1-7 Genomic Library Promoters Introns Intergenic Non-expressed genes cDNA Library Expressed genes Transcription start sites Open reading frames (ORFs) Splice points Slide 34 Purification of mRNA p. 1-8 Collect and grind up plants in mild denaturing solution Spin out debris (Tissue, membranes, etc) Treat with DNAse (removes DNA) Treat with Phenol (removes protein) Slide 35 Synthesis of cDNA from mRNA p. 1-8 Slide 36 SfiI digestion sites of pTRiplEX2 p. 1-9 Slide 37 p. 1-10 Cloning Duckweed cDNA fragments into the pTriplEX2 polylinker cDNA Insert Slide 38 WSSP-14 Chapter 1B Plasmid Preps Slide 39 Grow an overnight (ON) culture of the desired bacteria in 2 ml of LB medium containing the ampicillin antibiotic for plasmid selection. Incubate the cultures at 37C with vigorous shaking. 1. Grow the bacteria p. 2-11 amp Slide 40 20AV12.14 Naming your clones YearYour initials School #Clone # # School 01. Bayonne HS, NJ 02. Bridgewater HS, NJ 04. East Brunswick HS, NJ 05. High Point HS, NJ 06. Hillsborough HS, NJ 07. James Caldwell HS, NJ 09. JP Stevens HS, NJ 11. Montville HS, NJ 13. Pascack Hills HS, NJ 14. Pascack Valley HS, NJ 15. Rutgers Prep., NJ 16. Somerville HS, NJ 17. The Pingry School, NJ 18. Watchung Hills HS, NJ 19. West Windsor-Plains. HSS, NJ 38. Hackettstown, NJ 47. Fairlawn NH, NJ 49. Piscataway, NJ 50. The Frisch School, NJ 70. Old Bridge HS, NJ 93. The Peddie School, NJ 94. Academy of Edison, NJ 95. Acad. Of Enrichment & Adv., NJ 96. Holmdel HS, NJ 97. Robbinsville HS, NJ 98. Union City HS, NJ 103. The Hun School, NJ 104. Elmwood Park Memorital, NJ 105 North Brunswick, NJ # School 34. Science & Math Acad. MD 35. Walter Johnson, MD 59. Col. Zadok Magruder HS, MD 62. Winston Churchill, MD 81. South River HS, MD 82. Southern HS, MD 87. Kent Co HS IBALC, MD 88. Randallstown HS, MD 89. Gilman School, MD 100. Cristo Rey Jesuit HS, MD 101. Pikesville HS, MD 102. New Town HS, MD # School 65. Dougherty HS, CA 66. Modesto HS, CA 67. Tracy HS, CA 68. Waipuhu HS, HI 90. Granada High School, CA 91. Amador Valley High School, CA Slide 41 Enter the names of the clones into your schools Google Docs Clone Report sheet Slide 42 The average ON contains 10 9 cells/ml. Slide 43 Grown ON on the bench at RT Grown ON shaking at 37C Slide 44 One way to tell if your ON is fully grown is to see if you can see writing if you hold the tube up to your notes Fully Grown Needs to grow longer Slide 45 2a. Transfer the cells to a tube and centrifuge Transfer 1.5 ml of the culture to a microfuge tube and pellet the cells for 1 minute at full speed (12,000 rpm) in the microcentrifuge. First tap or gently vortex the glass culture tube to resuspend the cells which have settled. The culture can be transferred to the microfuge tube by pouring. p. 1-12 (Follow steps in Lab 6) Slide 46 2a. Centrifuge the samples Balance the tubes in the centrifuge Pellet the cells for 1 minute at full speed (10,000-14,000 rpm) in the microcentrifuge. Slide 47 2a. Centrifuge the samples Make sure there is a good size pellet Before After Slide 48 2b. Remove the supernatant Remove the growth medium (supernatant) by pouring out into a waste cup. Leave the bacterial pellet as dry as possible so that additional solutions are not diluted. Slide 49 3a. Resuspend the cell pellet Resuspend the bacterial pellet in 200 l of Solution I by pipeting up and down. Add 200 l of Solution I, cap the tube, and vortex on the highest setting (pipetman can be used). Look very closely for any undispersed pellet before proceeding to the next step. It is essential that the pellet be completely dispersed. Solution I contains three essential components: glucose, Tris and EDTA. Glucose and Tris are used to buffer the pH of the cell suspension. EDTA is a chemical that chelates divalent cations (ions with charges of +2) in the suspension, such as Mg ++. This helps break down the cell membrane and inactivate intracellular enzymes. p. 1-12 Slide 50 3. Resuspend the cell pellet in Soln. I Resuspend the bacterial pellet in 200 l of Solution I by pipeting up and down. Slide 51 3. Resuspend the cell pellet in Soln. I Make sure the pellet is fully resuspended! Not fully suspended Fully suspended Slide 52 3b. Store the suspended the cell pellet in you schools box at -20C It may take more than two weeks to perform the PCR and run the gel before you are ready to perform the miniprep. If the bacterial culture is left in the refridgerator then during this time many of the cells will die and the plasmid yeilds will go down. NEW CHANGE IN PROTOCOL!! Slide 53 4. Add Solution II Add 200 l of Solution II (0.2 N NaOH, 1%SDS), mix gently 4-6 times. Do not vortex!! This will shear the DNA and contaminate your DNA preps. Denatures protein, DNA, RNA, membranes. During this step a viscous bacterial lysate forms (the cells lyse). p. 1-13 Slide 54 4. Add Solution II (cont.) The cell solution should become clear Before After Slide 55 5. Add Solution III Add 400 l of Solution III (3 M KOAc, pH 4.8). Mix gently 4-6 times. Do not vortex. Solution III neutralizes cell suspension. A white precipitate consisting of aggregated chromosomal DNA, RNA and cell debris and SDS will form. Plasmids will renature p. 1-13 Slide 56 5. Add Solution III (cont.) Before Inverting Inverting 3 times Inverting 6 times White precipitate Slide 57 6. Centrifuge cell debris Centrifuge for 5 minutes at full speed in the microcentrifuge. A white pellet will form on the bottom and side of the tube after centrifugation. Slide 58 7. Transfer sup. (DNA) to spin column. Pour the supernatant to the appropriately labeled spin column which has been inserted into the 2 ml microcentrifuge tube. Slide 59 7. Transfer sup. (DNA) to spin column. Before After Slide 60 8. Centrifuge the spin column Centrifuge for 1 minute at full speed. Before After Slide 61 8. Centrifuge the spin column (cont.) Pour the flow-through from the collection tube. Slide 62 9. Wash the column with Wash Buffer Add 400 l of Wash buffer to the spin column contained in the 2 ml Collection Tube, centrifuge at full speed for 1 minute, and drain the flow through. This buffer helps to further remove any nucleases that may have co-purified with the DNA. Remove the liquid that has passed through the column in the same way as performed in Step 8. p. 1-14 Slide 63 9. Wash the column with Wash Buffer Spin Before After Slide 64 10. Spin the column a second time to remove all the Wash buffer Centrifuge again for 1 minute at full speed to remove any residual wash solution that might still be in the column. Any residual wash solution must be removed because the ethanol contained in this solution may interfere with further DNA manipulations. p. 1-15 Slide 65 11. Elute the DNA with EB Place the spin column into an appropriately labeled 1.7 ml microcentrifuge tube and add 60 ul of EB buffer to the column. Elutes the plasmid DNA from the column and collects in the microcentrifuge tube. p. 1-15 Slide 66 11. Elute the DNA with EB (cont.) p. 1-15 Centrifuge at full speed for 1 minute. Slide 67 12. Store your DNA p. 1-15 Remove the spin column from the labeled 1.7 ml microcentrifuge tube and close the lid on the tube tightly. Store the miniprep DNA in your freezer box (-20C).