composition of reagents in mol bio

8/22/2019 Composition of Reagents in Mol Bio

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Common Solutions For Molecular Biology Techniques.MOLARITY CALCULATOR

Molar/Molarity

Protocol to ship and reconstitute the plasmid cDNA.

Autoclave- Sterilization

Making LB Media

Making Bacterial Plates

Standard concentration of antibiotic of agar plates

Inter conversions of ng and pmol of oligos

Spectrophotometer reading and molecular weights

Making Competent CellsEasy Pure Midi-Mini Plasmid Preparation

Midi Plasmid Preparation (without kit) using silica resin:

Midi Plasmid Preparation (without kit) using phenol/chloroform:

Mini Plasmid Preparation (without kit) using phenol/chloroform:

Easy Pure Maxi/Mega Plasmid Preparation Kit

Large Scale Cesium Chloride Plasmid Preparation (without kit):

Easy clean DNA Spin Filters

Standard Conditions for PCR ProtocolPCR Purification

Genomic DNA Isolation (Solid Tissues)

Common Solutions For Molecular BiologyTechniques Ampicillin 50 mg/liter (for LB media) 100 mg/liter(for LB Agar Plates)

Ammonium persulfate (10%), 10 ml Dissolve 1 g ammonium persulfate(APS) in H2O and adjust the final volume to 10 ml. Always use freshsolution. Acryl-bisacrylamide mix (30%), 100 ml Dissolve 29 g acrylamide and 1g N,N-methylenebisacrylamide in H2O. Adjust the final volume to 100 ml.Store solution in brown bottle at 4C. Note: Acrylamide is a toxic and
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carcinogen. Use gloves and a mask when making the solution.

Agar plates: Use LB media or YT media except that add 15 g Bacto-Agar/liter. Sterilize by autoclaving. Allow the medium to cool (do not use icebuckets) to 45C and add the appropriate amount of antibiotic. Gently mixthe media by swirling and pour into petri plates. Allow plates to solidify and

store at 4C. ATP (0.1M), 1ml Dissolve 60 mg in 0.8 ml of water. Adjust pH to 7.0 with0.1 N NaOH. Make final volume to 1 ml and dispense solution to smallaliquots.

Ammoium Acetate (10M), 100 ml Dissolve 77.1 g in 100 ml of waterAcrylamide (30 %), 1 literDissolve 380 g of Acrylamide and 20 g of N,N-methylenebisacrylamide intotal volume of 1 L

Acrylamide (40 %), 1literDissolve 380 g of Acrylamide and 20 g of N,N-methylenebisacrylamide intotal volume of 600 mlBlocking solution Make a solution of 2.5 to 5% (w/v) nonfat dried milk inTBS or PBS. Bromophenol Blue (10%), 50 ml Dissolve 5 g of Bromophenol Blue in 50ml of sterile TE and store at room temperature. Chloramphenicol Dissolve in ethanol Make stock as 175 mg/ml ethanoland store at 20C. (For LB media or LB plates use up to 170 mg/liter) CaCl2 (0.1 M), 1 liter Dissolve 14.7 g of CaCl2.2H20 in 1 L water.Autoclave to sterilize. Filter through 0.22-micron filter. Store at roomtemperature.

CaCl2 (1 M) Dissolve 219.08 g calcium chloride -6H2O (MW = 219.08) inH2O. Adjust final volume to Sterilize by autoclaving and store at roomtemperature. Coomassie Blue staining solution, I liter Mix 2.5 g of Coomassie Blue(Coomassie Brilliant Blue R-250), 450 ml methanol, 100 ml acetic acid and450 ml H2O. Mix thoroughly and store solution at room temperature.

Coomassie Blue destaining solution, I liter Mix 450 ml methanol, 100ml acetic acid and 450 ml H2O. Mix thoroughly and store solution at roomtemperature.

Denhardt Solution (250 x) -1 liter. Dissolve 50 g Ficoll 400, 50 gpolyvinylpyrrolidone and 50 g bovine serum albumin (BSA) in 600 ml distilledH2O. Bring final volume to 1 liter with H2O. Store 25 ml aliquots at 20C.


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Denhardt Solution (50-X), 500 ml Add 5 g of polyvenylpyrrolidone(PVP), 5 g Bovine Serum Albumin (BSA), 5 g Ficoll Make final volume to 500ml. Sterilize filter and store at 4 degree C.

DTT (1M), 20 ml Dissolve 3.09 g of DTT in 20 ml of sterile water. Dispenseinto small aliquots and store at 20 degree C.

DTT (1M ), 10 ml Dissolve 1.54 g 1,4-dithio-DL-threitol (MW = 154.25) in10 ml 10 mM sodium acetate (pH 5.2). Store 1 ml aliquots at 20C. DEPC-treated water Add 0.2 ml diethylpyrocarbonate (DEPC) to 100 ml ofa water. Incubate overnight. Autoclave the solution and make aliquots. StoreDEPC treated water at room temperature.

EDTA (0.5 M Na2EDTA), pH 8.0, 1 liter Dissolve 186.12 g disodiumethylenediaminetetraacetate - 2H2O (FW: 372.24) in 800 ml H2O. Add ~20g NaOH pellets and stir vigorously on a magnetic stirrer for about 1 hour.Bring final volume to 1 liter with H2O. Ethidium Bromide (DNA Stain solution): Prepare a stock solution of 10mg/ml in sterile water. Stir until the dye has complete dissolved. Store at4C in a amber or brown bottle.

During electrophoresis: add 10 ul of stock solution per 100 ml of gel volume. Caution: Ethidium bromide is a mutagen and toxic. Wear gloves whilehandling Ethidium bromide. Electrophoresis Gel Loading Buffer (100 ml) Dissolve 250 mgbromophenol blue and 250 mg xylene cyanol in 30 ml 100 mM Tris pH 7.4.Add 70 ml glycerol and mix by vortexing. Store aliquots at roomtemperature.

Electrophoresis Gel Loading Buffer, 10 ml Glycerol 5 ml, 50X TAE 0.25ml, Bromophenol Blue 1ml from 1% solution, Xylene cyanol 1ml from 1%solution

IPTG (0.1 M), 50 ml Dissolve 0.595g isopropyl -D-thiogalactopyranoside(MW = 238.3) in H2O. Adjust final volume to 25 ml with sterile H2O. Make 1ml or 5 ml aliquots and sterilize by 0.22 m filtration and store at 20C.

IPTG. (100mM), 1 ml Dissolve 23.8 mg in 1 ml of sterile water and storeat 20 degree C(FW: 238.3)

Kanamycin 25 mg/ml in H2O and store at 20C. (For LB media or LBplates use 25 mg/liter)

LB Media: Mix 10 g Bacto-Tryptone, 5 g Bacto-Yeast extract and 10 g NaClin 900 ml H2O. Adjust the pH to 7.0 with approximately 0.2 ml of 10 MNaOH. Bring the final volume to 1 liter with H2O Sterilize by autoclaving.


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M9 minimal media: Mix 12.8 g Na2HPO4-7H2O, 3 g KH2PO4, 0.5 g NaCland 1 g NH4Clin 900 ml H2O. Adjust the pH to 7.4 with 10 M NaOH. Adjustthe final volume to 1 liter with H2O. Sterilize the solution by autoclaving.Once media is cooled, add 2 ml 1 M MgSO4 -7H2O, 0.1 ml of 1M CaCl2 and10 ml 20% glucose. Sterilize by filtering through 0.22 m filter. Store at 4C

MgCl2 (1 M), 1 liter Dissolve 203.31 g MgCl2-6H2O (MW = 203.31) in800 ml H2O. Adjust final volume to 1 liter and store at roomtemperature. MgCl2 (0.1M), 1 liter Dissolve 20.3 g mf MgCl2.6H2O in 1l water.Autoclave or use 0.22 micron filter.

MOPS (10 x), 1 liter Add 41.85 g 4-morpholinepropanesulfonic acid (MW= 209.27), 6.80 g sodium acetate-3H2O (MW = 136.08), 20 ml of 0.5 MNa2EDTA. Adjust pH to 7.0 with NaOH. Adjust final volume to 1 liter andstore aliquots in brown bottle at 4C.

NaOAc (3 M), 1 liter Dissolve 408.24 g sodium acetate -3H2O (MW =136.08). Adjust to required pH (4.0 or 5.2 or 7.0) with glacial acetic acid.Adjust final volume to 1 liter and store aliquots at room temperature.

NaCl (5M), 1 liter Dissolve 292.2 g sodium chloride (MW = 58.44) in H2O.Adjust final volume to 1 liter, autoclave and store aliquots at roomtemperature.

NH4OAc (10M) Dissolve 770.8 g ammonium acetate (MW = 77.08) inH2O. Adjust final volume to 1 liter. PBS (10 x), 1 liter Dissolve 80 g NaCl, 2 g KCl, 26.8 g Na2HPO4-7H2Oand 2.4 g KH2PO4 in 900 ml H2O. Adjust to pH 7.4 with 1M HCl. Adjust finalvolume to 1 liter, autoclave and store at room temperature.

Phenol, 1 liter Dissolve 500 g phenol in 250 ml 1 M Tris (pH 8.0). Removeupper aqueous phase and add 250 ml of 0.1M Tris (pH 8.0). Remove upperaqueous phase and add 250 ml of TE buffer. Store phenil soltuon in brownbottle at 4C. Potassium acetate (0.2 M), 1 liter. Dissolve 19.62 g potassium acetate(MW = 98.14) in 900 ml H2O. Adjust final volume to 1 liter, autoclave andstore at room temperature. RNA Sample buffer Mix 20 ml deionized formamide, 7 ml 37%formaldehyde and 4 ml 5 x MOPS. Note: Use gloves and fumehood.RNA Loading Buffer for Gel electrophoresis Mix RNA and loading buffer with3:1 ratio. Make stock solution of loading buffer as follows: 60% glycerol, 1mM Na2EDTA, and 0.5% Bromophenol Blue. Aliquots of stock solutions canbe store at 20C.


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Salmon Sperm DNA (5 mg/ml), 20 ml Cut 100 mg of Salmon SpermDNA with scissors to small pieces and dissolve in 20 ml of water. Stir thesolution by using magnetic stirrer for about 4 hours. Shear the DNA bypassing through a 17-gauge needle. Dispense DNA to small aliquots andstore at 20 degree C.

SDS PAGE (2X), 100 ml (Sample buffer) Add 20 ml 1.5 M Tris (pH 6.8),12 ml 20% SDS, 60 ml glycerol, 30 ml -mercaptoethanol and 3.6 mgbromophenol blue and adjust the final volume to 100 ml with H2O. makealiquots and store them at 20C. 4 SDS PAGE (10 X), 1 liter (Running buffer) Dissolve 10 g SDS, 30.3 gTris and 144.1 g glycine in H2O and bring the final volume to 1 liter. Store at

room temperature.

SDS (10%), 1 liter Dissolve 100 g sodium dodecyl sulfate powder in 900ml H2O. If necessary, warm the solution to dissolve. Adjust final volume to1 liter and store aliquots at room temperature. SOB: Mix 20 g Bacto Tryptone, 5 g Bacto yeast extract, 0.5 g NaCland 2.5 ml 1 M KCl in 900 ml H2O. Adjust pH to 7.0 with NaOH and bringfinal volume to 1 liter. Sterilize the solution by autoclaving. Before usingmedia add, add 10 ml filtered/sterile 1 M MgCl2. 4 SOC: Same as SOB mediaexcept that it contains 20 ml sterile 1 M glucose

Sodium acetate (0.2 M), 1 liter. Dissolve 27.21 g sodium acetate -3H2O(MW = 136.08) in 900 ml H2O. Adjust final volume to 1 liter, autoclave andstore at room temperature.

Sodium Phosphate (0.2 M) (mono-sodium), 1 liter

Dissolve 27.6 gNaH2PO4 -1H2O (MW = 138) in H2O, adjust final volume to 1 liter and storeat room temperature. Sodium Phosphate (0.2 M) (di-sodium), 1 liter Dissolve 53.62 gNa2HPO4-7H2O (MW = 268.1) in H2O,adjust final volume to 1 liter and storeat room temperature.

SYBR Green (DNA Stain solution) Stock solution is supplied in DMSO,which stable for about one year if stored at - 20C. Working solution: dilutethe stock solution 1:10,000 in gel buffer. Sensitivity: as low as 50 pg perband dsDNA SSC (20 x), 1 liter Dissolve 175.3 g NaCl and 88.2 gsodium citrate -2H2O in 900 ml H2O. Adjust pH to 7.0 with 1M HCl. Adjustfinal volume to 1 liter, autoclave and store at room temperature.

SSPE (20 x), 1 liter Dissolve 175.3 g NaCl, 27.6 g NaH2PO4 - 1H2O and7.4 g Na2EDTA in 900 ml H2O. Adjust pH to 7.4 with NaOH pellets or addabout 6.5 ml of a 10 M NaOH solution. Adjust final volume to 1 liter,autoclave and store at room temperature.

TAE (50X) 242 g Tris base 57.1 g glacial acetic acid 100 ml of 0.5 M


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EDTA (pH 8.0) Final volume 1LTBE (10X), I liter 54 g Tris base 27.5 g boric acid 20 ml of 0.5 M EDTA(pH 8.0) Final volume 1LTris-Glycine-SDS(5X), I liter 15.1 g Tris base 94 g Glycine(electrophjoreis grade, pH 8.3) 50 ml of 10% SDAS Final volume 1LTCA, 100% (w/v) Add 1 Kg trichloroacetic acid (TCA) to 454 ml H2O tomake 100% (w/v) TCA.

Tris (1 M), 1 liter Dissolve 121.14 g Tris base (hydroxymethyl) aminomethane (MW = 121.14) in H2O. Stir the solution with magnetic stirrer andadjust pH by adding concentrated HCl (7.4: ~ 70 ml, 7.6: ~ 60 ml, 8.0: ~ 42ml) Adjust final volume to 1 liter, autoclave and store at room temperature.

Tris-Glycine-SDS(5X), 1liter Add 15.1 g Tris base, 94 g Glycine(electrophjoreis grade, pH 8.3), 50 ml of 10% SDS. Bring final volume to 1L

TE (1 x ) 1 liter Add 10 ml 1 M Tris (pH 8.0, 7.6 or 7.4) and 200 l of 0.5M Na2EDTA to 900 ml H2O. Adjust final volume to 1 liter, autoclave andstore at room temperature.

TBE (10X), 1 liter Add 108 g Tris, 55 g Boric acid and 40 ml 0.5 MNa2EDTA in water. Adjust the final volume to 1 liter with H2O and Store atroom temperature.

TAE (50X), 1 liter Dissolve 242 g Tris in 800 ml H2O. Add 57.1 ml glacialacetic acid and 100 ml 0.5 M Na2EDTA. Adjust the final volume to 1 liter withH2O and Store at room temperature.

TPE (10X)- Tris-phosphate, 1 liter Mix 108 g Tris 15.5 ml 85%Phosphoric acid (1.679 g/ml) and 40 ml 0.5 M Na2EDTA in water. Adjustthe final volume to 1 liter with H2O and Store at room temperature.

Transfer Buffer (1X), 1 liter (For wet blots) Dissolve 2.9 g Glycine, 5.8g Tris and 0.37 g SDS in 0.2L methanol. Add water and adjust fnal volume to1liter. Store solution at 4C

TBS (1X) , I liter (Tris Buffered Saline) Dissolve 6.05 g Tris (50 mM) and8.76 g NaCl (150 mM) in H2O. Adjust pH to 7.4 with 1 M HCl (~ 9.5 ml) andadjust the final volume to 1 liter with distilled H2O. Store solution at 4C.

TBST (1X), 1 liter (Tris Buffered Saline + Tween) Dissolve 1 ml Tween20 in 1 liter TBS buffer. Top agar Plates: Use LB media or YT media except that add 7.5 g Bacto-Agar/liter. Sterilize by autoclaving. Allow the medium to cool (do not use icebuckets) to 45C and add the appropriate amount of antibiotic. Gently mixthe media by swirling and pour into petri plates. Allow plates to solidify and


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store at 4C.

X-gal/IPTG plate Before pouring the plates, add 1 ml of 0.2M IPTG and 1ml of X-gal (40 mg/ml) solutions. Final concentration of IPTG is 0.2 mM andX-gal is 40 g/ml X-gal (20 mg/ml), 10 ml Dissolve 200 mg 5-Bromo-4-Chloro-3-Indolyl-D-galactoside (X-gal) in 10 ml N,N-dimethyl formamide. Make 1 ml aliquotsand store at 20C. YT Media: Mix 8 g Bacto-Tryptone, 5 g Bacto-Yeast extract and 2.5 g NaClin 900 ml H2O. Adjust pH to 7.0 with approximately 0.2 ml 10 M NaOH.Adjust the final volume volume to 1 liter. Sterilize by autoclaving. and storeat room temperature. sterilize by autoclaving. Store at room temperature.

Molar/Molarity Example: How to make up a 50 ml of 5 M NaCl Formula: 5 mol/liter X 58.44 (FW) g/mol X 0.05 liter = 14.61 g.

Dissolve 14.61 g NaCL in water (final volume 50 ml) Note: Weight of 1 mol = 6. 023 x 1023 (Avogadros number) parts of amolecules.

Protocol to ship and reconstitute the plasmid cDNA.

Mark a circled area with a pen on a clean Whatman #1 filter paper. Spot

about 2 g of plasmid DNA. Allow the filter paper dry at roomtemperature. Insert spotted filter paper inside a poly bag and seal it properly

by using a thermal poly sealer.

Store clones (spotted on filter papers) at 4C until you are ready to use them.Clones are supplied as DNA (approximately 2 g) spotted onto Whatman #1

filter.

To recover the DNA, use clean gloves and cut the marked circle area that

contains dried plasmid DNA. By using clean forceps, insert the filter paperinto a 1.5 ml micro centrifuge tube. Add 100 l of TE buffer (10 mM

TRIS base, 1 mM EDTA, pH 8.0) to the micro centrifuge tube, vortexbriefly and incubate at room temperature for 5 minutes. Vortex again and

Centrifuge the tube for a few seconds and then remove about 10 l of

supernatant for use in transfecting E. coli by electroporation or chemicalmeans. Routine competent cells like HB101 or DH-5 alpha can be used for


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transformation. Pick up a single colony for making plasmid preparation

Please do not try to use the DNA directly for any application other than to

transform bacteria and prepare a plasmid stock.

Autoclave- Sterilization time: Usually 15 minutes to 30 minutesat 121C. Caution: certain buffers like SDS or organic solvents should not beautoclaved. To avoid bacterial contamination, some solutions may be filteredthrough 0.22 m filter.

Making LB Media: Weight 25 g LB broth powder in a 2 L flask andadd water to a final volume of 1L. Shake the contents and autoclave mediafor 30 minutes (liquid cycle). Make sure to add enough water (about 1L) in atray which contains your flask containing LB media. Add appropriateantibiotic after the media is cooled down (below 45 degree C).

Standard concentration of antibiotic of media: Ampicillin Plates 40 mg/L Kenamycin Plates 25 mg/L Chloramphenicol - 25 mg/L Streptomycin 25 mg/L Tetracycline 10 mg/L Making Bacterial Plates: Weight 37g LB broth Agar powder in a 2L flask and add water to a final volume of 1L. Shake the contents andautoclave media for 30 minutes (liquid cycle). Make sure to add enoughwater (about 1L) in a tray which contains your flask containing LB media.Allow flask to cool at room temperature. Do not use ice for cooling hot mediaotherwise media will solidify unevenly inside the flask. Once the temperaturecools down to about 50 degree C, add appropriate antibiotic. Shake the flaskthoroughly to mix the antibiotic.

Pour media into plates and immediately cover plates with lids. In order to dryup any excess of moisture, leave the plates overnight at room temperature.

The following day, store the plates upside down in a cold room orrefrigerator. Plates are stable and can be used up to 6 months.

Standard concentration of antibiotic of agar plates:

Ampicillin Plates 75 mg/L


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Kenamycin Plates 25 mg/L Chloramphenicol - 25 mg/L Streptomycin - 30 mg/L Tetracycline 15 mg/L

Inter conversions of ng and pmol of oligos. Spectrophotometer reading andmolecular weights.

10 pmol = 3.3 ng X n, where n in the number of nucleotides in a primer. 1 ug of 1000 bp DNA = 1.52 pmol DNA (double stranded)Molecular weight= number of bases X 650 I kb DNA = ~ 333 amino acids = 37K Dalton protein 270 bp DNA = 70 amino acids = 10K Dalton protein.

I OD (at 260 nm) = 50 g/ml of ds DNA I OD (at 260 nm) = 33 g/ml of ss DNA I OD (at 260 nm) = 40 g/ml of ds RNA

Inter conversions of ng and pmol of oligos.

10 pmol = 3.3 ng X n, where n in the number of nucleotides in a primer.1 ug of 1000 bp DNA = 1.52 pmol

Spectrophotometer reading and molecular weights.DNA (double stranded) molecular weight= number of bases X 650 I kb DNA = ~ 333 amino acids = 37K Dalton protein 270 bp DNA = 70 amino acids = 10K Dalton protein. I OD (at 260 nm) = 50 g/ml of ds DNA I OD (at 260 nm)= 33 g/ml of ss DNA I OD (at 260 nm) = 40 g/ml of dsRNA

Making Competent Cells

Day 1.

1. Autoclave 250 ml capacity centrifuge bottles. This will be used on Day 2.

2. Autoclave 1L of LB broth media in ~2L capacity flask and keep media at


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4C until next day.

3. Streak culture of cells on a LB plate (without antibiotic), and incubate itovernight to get isolated colonies of E. coli cells. Alternatively, you can growabout 100 l of cells (fresh or frozen stock) in about 15 ml LB media (withoutantibiotic) and incubate overnight at 37C by shaking. It is recommended

to have a control- as separate LB plate or LB media tube (without antibiotic)for simultaneous growth, to ensure that no growth is visible in control tube.

Day 2:

1. Transfer a single colony or small amount of culture (~ 5ml) to 1 L ofsterile LB broth media. Note: Since E.coli cells do not have any antibioticresistance, do not add any antibiotic.

2. Grow cells on a shaker at 37C until media growth as an OD of 0.4 to0.5@ 600 nm you can use 1 cm pathlength cuvette to check the OD.

3. Use large capacity (250 ml) autoclaved centrifuge bottles to centrifugecells at 5,000 RPM for 10 minutes at 4oC. Discard the supernatant.

4. Make 100 mM MgCl2 and 100 mM CaCl2 solutions and keep them on ice atthis point. It is recommended to filter both solutions. Keep filtered solutionsin ice until further use.

5. Gently re-suspend the bacteria pellet with 1/10 volume of ice-cold MgCl2solution. If your original growth volume was 1L, add total of 100 ml of MgCl2solution in all bottles. Do not VORTEX cells. Centrifuge the cell suspension at4,000 RPM for 10 minutes at 4oC. Discard the supernatant.

6. Re-suspend the bacteria pellet with 1/20 volume of ice cold CaCl2solution. If your original growth volume was 1L, add total of 50 ml of MgCl2solution. Do not VORTEX cells. Keep this suspension on ice for at least 30minutes.

7. Centrifuge the cell suspension at 4,000 RPM for 10 minutes at 4C.Discard the solution and re-suspend the cell pellet in 1/50 volume of ice-coldCaCl2 solution. If your original growth volume was 1L, add 20 ml of CaCl2solution. Add 3.5 ml of glycerol and re-suspend the competent cells. Makesure to carry all steps at or below 4C. Dispense competent cells as 100 Laliquots and freeze at -80oC.

Midi Plasmid Preparation (without kit) using silicaresin:(To buy resin in bulk, please contact primmlabs.com)


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A midi-prep double-stranded DNA isolation provides 4-5 times amount oftemplate DNA as compared to plasmid made by protocols used for makingmini plasmid preparations. In this protocol, a single bacterial colony havingplasmid of interest is inoculated into 15 ml of liquid media containingappropriate antibiotic. The culture is incubated further for about 16 hours.After harvesting, the cells are centrifuged and silica-based alkaline-lysispurification is performed. The approximate yield of double-stranded DNAusing this method is approximately 1 g of DNA per ml of cell culture.

Protocol

1. Pick up a colony of bacteria containing the plasmid DNA of interest into 50ml capacity Falcon tube containing 15 ml of LB media supplemented with theappropriate antibiotic. Incubate culture with shaking at 37C for about 16hours.

2. Harvest the cells by centrifugation at 5000 rpm for 5 minutes and decantthe supernatant. The cell pellets can be frozen at -70C at this point.

3. Resuspend the cell pellets in 0.5 ml ofGTE (Glucose Tris EDTABuffer)/Lysozyme solution. Incubate the solution for 10 minutes.

4. Add 0.5 ml ofAlkaline Lysis Solution. Mix gently until solution clears.Leave the solution on ice for 10 minutes.

5. Add 0.5 ml ofAcetate solution. Mix gently, but avoid vortexing. Leavethe solution on ice for 10 minutes.

6. Transfer the solution to a microfuge tube and centrifuge at 10,000 rpm for20 minutes.

7. Decant the supernatant to a new microfuge tube and add 5 ul of a 20mg/ml DNase-free RNase A. Incubate sample in a 37C water bath for 10minutes.

8. Add 1.5 ml of silica resin and allow the DNA to bind at room temperaturefor 5 minutes with occasional mixing. Centrifuge the solution at 3,000 for 2minutes.

9. Decant the supernatant, and resuspend the pellet with 1.5 ml of 70%ethanol. Centrifuge the solution at 3,000 for 2 minutes. Repeat the washingstep one more time.

10. Decant the supernatant and dry resin pellet in a vacuum oven.

11. Resuspend the pellet with 300 l of TE buffer. Remove Silica resin bycentrifugation at 10,000 rpm for 5 minutes.


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12. Transfer the supernatant into a clean 1.5 ml microfuge tube. Ifnecessary, further purification could be carried out by ethanol precipitationProcedure for additional purification: Add 1/10 volume of 3M sodium acetate(pH 7.0) and 3 volumes of ethanol. Spin sample for 10 minutes at 10,000rmp. Wash the pellet with 70 ethanol, Resuspend the final plasmid DNA in300 l of TE buffer.

Buffers:

GTE (Glucose Tris EDTA Buffer):Glucose: 1.8 Grams(1M) Tris pH 8.0: 5.0 mL(0.5M) EDTA: 4.0 mLFinal Volume: 200 mLAutoclave and store solution at 4C

GTE/Lysozyme buffer:GTE Buffer: 38.0 mLLysozyme Cocktail: 2.0 ml

Lysozyme Cocktail:Tris (1 M pH 8.0): 3.0 mlSterile H2O: 9.0 mlLysozyme: 60 mg

Final Volume: 12 ml

Lysis Solution:NaOH ((10M): 8 mLSDS (10%): 80 mLFinal Volume: 800 mL

Acetate Solution:Pottasium acetate: 245.5 GramsGlacial acetic acid: 57 mLFinal Volume: 500 mL

******

Midi Plasmid Preparation (without kit) usingphenol/chloroform:

A midi-prep double-stranded DNA isolation provides 4-5 times amount oftemplate DNA as compared to plasmid made by protocols used for making


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mini plasmid preparations. In this protocol, a single bacterial colony havingplasmid of interest is inoculated into 15 ml of liquid media containingappropriate antibiotic. The culture is incubated further for about 16 hours.After harvesting, the cells are centrifuged and phenol/chloroform basedpurification is performed. The approximate yield of double-stranded DNAusing this method is approximately 1 g of DNA per ml of cell culture.

Protocol

1. Pick up a colony of bacteria containing the plasmid DNA of interest into 50ml capacity Falcon tube containing 15 ml of LB media supplemented with theappropriate antibiotic. Incubate culture with shaking at 37C for about 16hours.

2. Harvest the cells by centrifugation at 5000 rpm for 5 minutes and decantthe supernatant. The cell pellets can be frozen at -70C at this point.




6. Transfer the solution to a microfuge tube and centrifuge at 10,000 rpm for

20 minutes.


8. Add equal amount of phenol pH 7.4/ chloroform / isoamylalcohol(25:24:1) solution and vortex the DNA for 5 minutes to extract the DNA.Note: Use gloves and safety glasses while handling phenol and chloroform.

9. Spin the tube for 10 minutes at 10,000 rpm. Gently remove the topaqueous phase and transfer into a fresh microfuge tube. Add equal amount

of chloroform / isoamylalcohol (24:1) and vortex the solution for 5 minutes.

10. Spin the tube for 10 minutes at 10,000 rpm. Gently remove the topaqueous phase and transfer into a fresh microfuge tube. Add 1/10 volume of3M sodium acetate (pH 7.0) and 3 volumes of ethanol.


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11. Spin sample for 10 minutes at 10,000 rmp. Remove supernatant andpulse in the microfuge and remove the remainder of the liquid. Resuspendthe final plasmid DNA in 300 l of TE buffer.

Buffers:


GTE/Lysozyme buffer:GTE Buffer: 38.0 mLLysozyme Cocktail: 2.0 ml

Lysozyme Cocktail:Tris (1 M pH 8.0): 3.0 mlSterile H2O: 9.0 mlLysozyme: 60 mgFinal Volume: 12 ml

Lysis Solution:NaOH ((10M): 8 mLSDS (10%): 80 mLFinal Volume: 800 mL


******

Mini Plasmid Preparation (without kit) using

phenol/chloroform:A mini-prep double-stranded DNA isolation provides small amount oftemplate DNA suitable for DNA sequencing or restriction enzyme digestion. .In this protocol, a single bacterial colony having plasmid of interest isinoculated into 5 ml of liquid media containing appropriate antibiotic. Theculture is incubated further for about 16 hours. After harvesting, the cellsare successively incubated with lysis buffer, alkaline detergent, and sodium


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acetate. Following lysis, solution is centrifuged and phenol/chloroform basedpurification is performed. The collected DNA is concentrated by ethanolprecipitation. The approximate yield of double-stranded DNA using thismethod is approximately 1 g of DNA per ml of cell culture.

1. Transfer 1.5 ml of overnight bacterial culture into a separate microfugetube and centrifuge for 2 minutes at 10,000 rpm. Remove the clearsupernatant. Add a further 1.5 ml of bacterial culture to the tube. Spin andremove the supernatant as before. Pellet represents 1.5 ml of culture.




5. Transfer the solution to a microfuge tube and centrifuge at 10,000 rpm for10 minutes.


7. Add an equal volume of Phenol. Note: stock phenol will have two phases,

the bottom phase is the phenol. Vortex for 30 seconds to 1 minute.

8. Spin samples for 3 minutes at 10, 000 rpm and remove the upperaqueous layer to a fresh tube. There will be two distinct layers with aninterface that may have white denatured proteins. Carefully remove theupper aqueous layer without disturbing the denatured protein or phenollayer.

9 . Spin the tube for 5 minutes at 10,000 rpm. Gently remove the topaqueous phase and transfer into a fresh microfuge tube. Add equal amountof chloroform / isoamylalcohol (24:1) and vortex the solution for 5 minutes.

10. Spin the tube for 10 minutes at 10,000 rpm. Gently remove the topaqueous phase and transfer into a fresh microfuge tube. Add 1/10 volume of3M sodium acetate (pH 7.0) and 3 volumes of ethanol. Place the tube at -20oC or at -70oC for 20 minutes.


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11. Spin sample for 10 minutes at 10,000 rmp. Remove supernatant andpulse in the microfuge and remove the remainder of the liquid. Resuspendthe final plasmid DNA in 100 l of TE buffer.

Easy Pure Midi-Mini Plasmid Preparation

Catalog: P-5050

This kit is good for purifying plasmid preps for routine analytical analysis,restriction digestion, PCR reactions and including transfection. Reagents areenough for about 75 midi preps. Midi preps for following protocol will takealmost same time as mini preps. Also, there is no significant cost differencein growth media for midi verses mini preps. Therefore, it is recommended touse this kit for making midi preps only-the yield will be sufficient for thescreening of positive colonies and for subsequent molecular biologyapplications. This protocol is designed to make midi plasmid preparations

which could give as high as ~75 g of high copy plasmid DNA. You can scaledown the procedure for mini preps.

CONTENTS:

Resuspension Solution: 30 ml Lysis Solution: 30 ml Neutralization Solution: 30 ml Precipitation Solution: 100 ml (in brown bottles) Ethanol wash: 20 ml (10X)

Complimentary Solutions:

1X TE (Tris EDTA Buffer) 30 ml 10 X TE (Tris EDTA Buffer) 30 ml Gel Running Buffer (50 X TAE) 50 ml (Running electrophoresis buffer: 0.5 X)

Midi Preps:

1. Grow ~10 ml bacterial culture for 12-15 hrs with vigorous shaking at 37C.50 ml capacity disposable polypropylene tubes usually work well for growthof 12 ml of bacterial cultures. Pellet the cells by centrifugation at 3000 rpm(2200g) for 15 minutes. Discard the supernatant. Spin the tube again for 1minute to remove residual media.

2. Re-suspend the pellet in 300 l ml of Re-suspension Solution. Vortex to


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re-suspend the pellet. The bacterial pellet should be re-suspended completelyuntil no cell clumps are visible. Let the cell suspension stand at roomtemperature for 5 minutes.

3. Add 300 l of Lysis Solution and mix the contents gently until the cellsuspension is clear. Let the solution stand for 5 minutes at room

temperature. Do not vortex the solution after adding Lysis Solution.

4. Add 300 l of Neutralization Solution and mix the contents by briefvortexing. Let the tube stand for 5 minutes at room temperature. Transferthe solution to a microfuge tube and spin at maximum speed for 5 minutes

5. Transfer the supernatant to a microfuge tube and spin at maximum speedfor 5 to 15 minutes.

6. In the mean time, dispense 1.2 ml of DNA Precipitation Solution into 2 mlof provided microfuge tubes.

7. Add 600 l of clear supernatant (step 5) into tube containing DNAPrecipitation solution. Vortex the solution and keep tubes in ice or at 20Cfor 10 minutes.

8. Spin tubes fro 20 minutes in cold room or use cold centrifuge.

9. Discard the solution and gently add ice-cold Ethanol Wash Solution (~1ml) discard it immediately. In order to avoid dislodging the pellet, it isrecommended to add ethanol wash from sides of the tube. Note: DNA pelletmay be too small and it may not be visible.

Ethanol Wash Solution: . Add 1 ml of Conc. DNA Wash solution+ 24 ml water

+ 25 ml Ethanol. Alternatively, add 20 ml of concentrated 10X ethanol washinto a clean bottle; add 180 ml water and 200 ml of ethanol. Keep bottlecontaining working Ethanol Wash Solution at 4C. Wash solution is stable for 1year.

10. Spin tubes for 5 minutes and remove any traces of ethanol wash with apipette tip.

11. Add 100 l of water or TE. Keep tubes at 37 to 42 degree C water bathfor about 10 minutes. Vortex tubes briefly to dissolve the DNA.

Mini Preps:

Scale down the solutions according to growth media. Protocol is same asabove except minor changes as below:

1. Avoid washing step (step # 9): after spinning the tube (step # 8), discardsolution and spin the tube again for about 5 minutes in cold room or use coldcentrifuge. Remove any traces of solution. 2. Add ~50 l of water or TE.Keep tubes at 37 to 42 degree C water bath for about 5 minutes. Vortex


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tubes briefly to dissolve the DNA.

-----------------------------------

Easy Pure Maxi/Mega plasmid preparation kit (20 Preparations)

Following protocol is designed for making maxi plasmid preparations, whichcan be used for most of molecular biology applications such as transfectionexperiments, cloning, restriction digestion or PCR. This kit provides reagentsfor 20 maxi plasmid preps. Important features of our Maxi plasmid isolationkit are:

* Extremely simple to perform. * You get up to 5 mg of DNA/-0.5L culture or ~95% of the amount present inthe bacterial pellet. * 260/280 ratio is ~1.8. * It is very inexpensive in comparison to other vendors. * Use Gloves while handling solutions for making plasmid preparations.

CONTENTS: Resuspension Solution: 200 ml Lysis Solution: 200 ml Neutralization Solution: 200 ml Suspension Solution: 20 ml Precipitation Solution A: 40 ml Precipitation Solution B: 20 ml Concentrated RNAse: 250 l 1X TE (Tris EDTA Buffer): 30 ml 10 X TE (Tris EDTA Buffer): 30 ml Gel Running Buffer (50 X TAE): 50 ml

Procedure:1. Grow ~ 500 ml of bacterial culture at 37C for 12-15 hrs with vigorousshaking. Pellet the cells by centrifugation at 5000 rpm for 10 minutes. Re-suspend the pellet in 10 ml of Re-suspension Solution. The pellet should be


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re-suspended thoroughly until no cell clumps are visible. Let the cellsuspension stand at room temperature for 5 minutes.

Note: Growth of 500 ml culture of high copy plasmid may contain as high as5 mg of plasmid DNA. Unlike column or resin based procedures, thisprocedure is designed to isolate entire amount of plasmid DNA from your

culture. Therefore, depending upon your needs, you may scale up or downyour preparations.

Suggestion:If a large amount of DNA prep is required but you do not wishto process at the same time, follow following simple steps: Grow media in1-liter culture. Re-suspend your pellet in 10 ml of TE buffer as a stock.Proceed with only 1/5th of stock pellet and save rest in a freezer (-20C) for

future use. Saving your stock of re-suspended pellet for future use will notaffect the quality of DNA. Take 2 ml of re-suspended pellet and add 8 ml ofRe-suspension solution. The rest of procedure is same.

2. Add 10 ml of Lysis Solution and mix the contents gently by inverting thetube 5-6 times. Let the solution stand for 5 minutes at RT. Do not use vortexto mix the contents.

3. Add 10 ml of Neutralization Solution and mix the contents by briefvortexing (5 seconds). Keep the solution in ice for about 10 minutes. Spinthe tube at 12, 000 rpm for 20 minutes. The supernatant at this step should

be clear with no visible clumps. Transfer the clear supernatant to a new tubeby using a cheese cloth.

Note: If solution is not fully transparent, some precipitates may have goneinto the filtered lysate. In that instance, spin the lysate for 10 minutes at

12,000 rpm and gently transfer the lysate to a new bottle.

4. Add 0.7 volume (21 ml) of Isopropanol to a bottle containing clear filtrate.Let the contents stand on ice for 5 minutes. Spin the tube for 10 minutes at12, 000 rpm. The temperature during spinning could be set anywhere from 4to 15C. Discard the supernatant completely and spin the tube again (2-5minutes) to remove any residual supernatant.

RNAse treatment

5. Dissolve the pellet completely in 0.7 ml of DNA Solublizing Solution. Add

10 l of concentrated RNAse that is DNAse free. Let the tube stand for 10minutes at room temperature. If the solution is not clear, spin the tube for 1minute at maximum speed to remove any insoluble impurities. Transfer theclear solution equally into two microfuge tubes (2 ml capacity) as provided.

DNA Purification 6. Add 1 ml of DNA Precipitation Solution A in each tube. Mix the contents by


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brief vortexing and incubates tubes at room temperature for at least 10minutes. Add 0.4 ml of Precipitation Solution B in each tube and vortex tubeto mix the contents. Centrifuge tubes in a cold centrifuge for 10 min atmaximum speed to precipitate the DNA.

Note: After incubation, it is safe keep the tubes in refrigerator (~4C) untilnext day.

7. Remove the supernatant; rinse the pellet gently with 80% ethanol. Makesure that do not loose the pellet. Spin the tubes briefly for few seconds andcarefully remove traces of ethanol wash without losing the DNA pellet. If youneed plasmid DNA which is absolutely free of any salt, follow optional step(below), otherwise dissolve the DNA by using 0.75 ml of 1X TE. At this pointthe DNA pellet may be quite compact but place tubes containing DNA in awater bath (~42C) for 10 minutes. Vortex or pipet the solution up and downto dissolve the DNA.

Optional step: Add 1 ml of 70 % ethanol and 100 l of 3M sodium acetate(provided in 2 ml tube) in a tube containing DNA pellet. Vortex the tubetubes rigorously for 30 seconds. Keep tube in ice for 5 minutes. Pellet theDNA by spinning tubes in a cold centrifuge for 10 min at maximum speed.Carefully remove ethanol wash. Note: Prefer to transfer the ethanol washinto a new tube to avoid accidentally throwing away the DNA pellet.

Note: Take the spectrophotometric reading of DNA in a TE buffer. Avoidtaking the reading in water as it can cause a shift in the OD due to an acidicpH of the water.

Large Scale Cesium Chloride Plasmid Preparation(without kit):The method used for the preparation of large-scale plasmid DNA by using analkaline lysis followed by ultracentrifugation. in cesium chloride-ethidiumbromide gradients. Briefly, grown culture containing the desired plasmid iscentrifuged, pellet is incubated in a lysozyme buffer, and treated withalkaline lysis reagents. Lysis buffer is used to solubilize proteins andmembranes are precipitated with low pH acetate buffer. Aftercentrifugation, the lysate is cleared by filtration through cheesecloth. ThePlasmid-containing supernatant is transferred to a new tube, and the plasmid

DNA is precipitated and the DNA pellet is resuspended in a buffer containingcesium chloride and ethidium bromide. DNA solution is loaded into specialplastic tubes and subjected to ultracentrifugation for about 16 hours. Highspeed centrifugation establishes cesium chloride density gradient, and theethidium bromide stained plasmid is visualized under long wave UV light.The lower band of super coiled plasmid DNA is removed by using a syringe.The dye, ethidium bromide is separated from the DNA by using salt saturated


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isobutanol. The final DNA pellet is resuspended in TE buffer and assayed byagarose gel electrophoresis.

1. Inoculate 10 ml LB (+ Antibiotic) with a single colony or 10 L offrozen glycerol stock and grow culture for about 16 hours or overnightat 37C.

2. Transfer 5 ml of the culture to 1 L of similar media and incubate for anadditional 8-10 hours or overnight with vigorous shaking at 37C.

3. Transfer the cells to 500 mL centrifuge bottles and spin at 5000 RPMfor 20 minutes.

4. Decant the supernatant, save the cell pellet. The cell pellets can befrozen at -70 C at this point.

5. Resuspend the cell pellet in 40 mL GTE/Lysozyme buffer. Gentlybreak the pellet with a pipette and incubate for solution for10 minutesat room temperature. Do not vortex the lysate as it may shear thechromosomal DNA.

6. Add 40 ml of Alkaline Lysis Solution. Mix gently until solutionclears. Leave the solution on ice for 15 minutes.

7. Add 40 ml of Acetate solution. Mix gently, but avoid vortexing.Leave the solution on ice for 15 minutes.

8. Transfer the solution into 250 ml centrifuge bottles and centrifuge at10,000 rpm for 30 minutes at 4C by using the GSA or equivalentrotor.

9. Filter the supernatant through cheesecloth into fresh centrifugebottles.

10.Add 90 ml of HPLC grade Isopropanol. Swirl to mix, and incubate inan ice-water bath for 30 minutes.

11.Collect the Isopropanol -precipitated DNA by centrifugation in 250 ml

bottles at 7000 rpm for 20 minutes at 4C.12.Carefully discard the supernatant and dissolve the pellet in a combinedtotal of 32 ml TE buffer, 5 ml of 5 mg/ml ethidium bromide, and 37 gcesium chloride. Final concentration of cesium chloride should be 1g/ml).

13.Transfer the sample into 35 ml plastic ultracentrifuge tubes, removeair bubbles, seal with rubber stoppers, and crimp properly. Note: UseTE to bring volume to just below the neck. Quick-Seal tubes (fromBeckman cat # 342412) can also be used.

14.Centrifuge overnight (16-20 hours) at 60,000 rpm at 15-20C in theSorvall OTD-75B ultracentrifuge (DuPont) using the T-865 or NVT90 orVTi80 rotor.

15.Carefully remove the centrifuge tubes from the rotor. And Place inclamp stand. Visualize the ethidium bromide stained DNA band underlong-wave UV light, and remove the lower DNA band using a 5 or 10cc syringe. Sometimes, two pink bands are too close to each otherand it may be helpful first to remove and discard the top band which ischromosomal DNA.

16.Transfer the plasmid DNA from syringe to 15 mL Falcon tube. Add 10mL of NaCl/water saturated n-butanol. Vortex the mixture and spin in


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a clinical centrifuge for 5 minutes.17.Remove top organic phase that contains pink colored ethidium

bromide solution and discard it at a safe place. Note: Ethidiumbromide is carcinogenic.

18.Repeat butanol extraction 3 to 5 times until the organic phasebecomes clear.

19.Transfer clear DNA solution to a clean JA17 centrifuge tube. Add 1/10 thvolume of 3M sodium acetate and 3 volumes of 100% ethanol.Precipitate at room temperature for 20 minutes.

20.Spin DNA at 10,000 RPM for 20 minutes in JA17 rotor at roomtemperature.

21.Discard supernatant and resuspend pellet in 2 ml of 0.3 M Na Ac pH5.2. Add 6 mL of 100% ethanol and spin DNA at 10,000 RPM for 20minutes.

22.Wash the pellet carefully with 70% ethanol. Dry the pellet usingSpeed-Vac and Resuspend the pellet in 500 l of TE by vortexing.

23.Store plasmid DNA in freezer.

Buffers:


GTE/Lysozyme buffer:

GTE Buffer: 38.0 mLLysozyme Cocktail: 2.0 ml

Lysozyme Cocktail:Tris (1 M pH 8.0): 3.0 mlSterile H2O: 9.0 mlLysozyme: 60 mgFinal Volume: 12 ml12 ml - enough for 6 samples, prepare 2 ml aliquots (fresh each time).

Lysis Solution:NaOH ((10M): 8 mL

SDS (10%): 80 mLFinal Volume: 800 mL



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NaCl/Water saturated n-Butanol:Mix equal volume of NaCl ((0.25 M) and n-Butanol. Votex the solution andallow the phases to separate. Use the upper organic phase (n-Butanol) forextractions.

EASY SPIN FILTER PROTOCOLS

Principle:Quick Spin columns contain gel filtration matrix which allow large molecules(e.g., DNA or RNA) to pass through quickly by molecular sieving whileretaining small contaminants such as salts and small nucleotides.

Protocol #1Protocol for the extraction of DNA fragments from high meltagarose gels.

(This protocol does not require gel filtration matrix)

1. Excise the gel slice from a high melt agarose containing the DNAband of interest. Slice gel into small pieces and place it inside the spinfilter. Insert the spin filter into receiving tube. This protocol is suitablefor high melt agarose gel only, do not use low melt agarose. We alsorecommend using 0.5 X TAE buffer for agarose gel electrophoresis.

Optional step: In order to increase the recovery of DNA, place the spinfilter (containing your gel slice) at -80 degree centigrade for at least 10

minutes. However, you can keep it at

80 degree centigrade forindefinite period of time which will not result in loss of quality of DNA.

2. Quickly, centrifuge for 5 minutes at maximum speed (12-14,000 g).

3. Receiving tube will contains eluted DNA which can be used directlyfor subsequent reactions such as labeling, DNA ligation, PCR, fill-in/kinase reaction with the Klenow fragment or four deoxynucleotidesand rATP sequencing reactions etc.,


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Note: Recovery of DNA is usually between 60 to 80% - depends uponsize and concentration of DNA. You may verify the DNA concentrationby using small aliquot of eluted DNA and run it on a gel.

If you are planning to use eluted DNA for DNA ligation, it isrecommended that you run the gel electrophoresis by using 0.5X TAEas running buffer. After extracting the DNA from gel slice, add an equalamount of water to the eluted DNA from the spin filter. Saltconcentration will be low enough and will not interfere with subsequentligation reactions.If you wish to remove TAE buffer completely or to concentrate DNA,follow protocol # 2. Do not directly concentrate eluted DNA (fromagarose gel slices) with ethanol as any trace of agarose will co-

precipitate with your DNA which will make it difficult to resuspend yourprecipitated DNA.


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Protocol #2Purification of DNA and removal of salt or unused primers or

dNTP's from the PCR/restriction enzyme products.

This protocol is applicable to following: Remove salts from enzymatic reaction products. Remove unused primers, dNTP's from the PCR prior to cyclesequencing Remove excess labels/ nucleotides from random or end labeling

reactions. Remove salts/buffers from eluted agarose gel slices (from protocol#1).

A: Hydrated Sephadex G-50:Weigh about 1 gram of Sephadex G-50

and suspend it in about 25 ml of sterile water. Sephadex is fully swelledin about 5 hours or leave overnight at room temperature. OnceSephadex is fully swelled, store it at 4 degree centigrade for furtheruse. It is recommended that you make few aliquots of hydrated gel inseparate tubes for further use. Hydrated gel with sufficient amount ofsterile water can be stored at 4 degrees C for about 6 months.

B: Loading gel into spin column and removal of excess fluid:

1. By using a pipet, remove most of water from the Sephadexcontaining tube. This should leave you with thick slurry of Sephadex G-

50. Add ~700 l of Sephadex G-50 slurry into a spin column. Insertspin column into a receiving tube.2. Make sure that the centrifuge is fully balanced. Spin for two minutes@ 2000 RPM and remove the excess fluid from the receiving tube.Optional: In order to tightly pack the column, you can refill the gel withadditional Sephadex G-50 slurry and spin once again for two minutes @2000 RPM. 3. Insert spin filter back intoreceiving tube and transfer 20 to 50l of the sample to the center of gelmatrix without disturbing the Sephadex bed surface. Centrifuge for 3minutes @ 3000 RPM. Store eluted DNA at -20 degree centigrade forsubsequent uses.

Note: Care must be taken to place the sample directly in the middle ofthe Sephadex gel surface of the column. Touching the sides of thecolumn may reduce the separation efficiency. Load the sample within10 minutes to avoid drying of the gel.


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Optional: Small amount of extracted DNA can be analyzed by agarosegel electrophoresis to verify the quantity of eluted DNA. If necessary,the remainder of the sample can be concentrated by ethanolprecipitation. In order to concentrate the DNA, add 1/10 volume of 3 Msodium acetate and 3 volumes of 100% ethanol. Keep the solution inice or at -20 degree for 10 minutes. To precipitate the DNA, Spinsamples for 10 minutes at maximum speed by using a cold microfuge.Remove traces of ethanol by using a clean pipette tip and resuspendthe DNA pellet in an appropriate amount of water or TE.

Standard Conditions for PCR Protocol

PCR is highly sensitive to contamination which can cause theamplification of unintended DNA bands or the reaction to fail with no

amplified product. Therefore, always use a positive control template,DNA that you know should amplify with primers. In order to avoid

cross-contamination, always use a clean tip every time you use acocktail and reagent or sample.

Most of the companies that provide thermostable enzyme also supply

buffer for PCR reactions. Optimum PCR product depends upontemplate and primers and salt concentration. However, the following

is a standard protocol for routine PCR:

For one 100 l reaction, add following in the sequence as shown below. Vortexes

PCR mix thoroughly after you add each component.

Ultra pure H20 85 l

10x PCR Buffer 10 l

dNTP (25 mM each dNTPs) 1 l

Primer 1 (add 40 p mol) 0.4 l (from stock of 100 pmol/l)

Primer 2 (add 40 p mol) 0.4 l (from stock of 100 pmol/l)


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DNA Template 1 l (~100 to 500 g)

Taq or Vent Pol 2 l (~4 Units)

Optional: Add 2.0 l 25 mM Mg Cl2 if PCR product yield is not enough.

Note: Thaw your DNA completely before using it as template. Keep all reagents

in ice. Always store dNTP at20 degree C. See page about calculations for

making stock solutions (100 pmol/l) of primers or inter conversions of ng and

pmol of oligos.

Standard program profile for PCR (~1 kb in size) is as follows:Step Temp C Time (minutes) cycles

1 95 (Denaturation) 5:00 12 95 (Denaturation) 1.00 (see step 5)

3 58 (Annealing) 1.00 (see step 5)

4 72 (Extension) 1.:00 (see step 5)

5 30 cycles steps 2 to 4.

6 72 (Extension) 15.00 1

7 END

Note: if you have a thermocycler which has a temperature gradient feature, make

PCR cocktail of about 300 l and run 6 reactions (50 l each) at different

annealing temperatures. Run 5 l of PCR product in a gel to find out the best

conditions.

PCR PurificationProtocol 1: Application: This protocol purifies and removes mostof contaminants from PCR product. This protocol will also denaturesthermostable enzyme which will not interfere during subsequent

reactions such as sequencing or restriction enzyme digestion of PCRproducts.

CONTENTS: PCR-DNA Purifier/Precipitation Soln. 30 ml Ethanolwash 20 ml (10X) TE (Tris EDTA Buffer) 30 ml 10 X TE (Tris EDTABuffer) 30 ml


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1. Transfer 100 l of PCR reaction mixture into a new microfuge tube.Add 300 l of PCR-DNA Purifier/Precipitation Solution. Vortex themixture and let the tube stand for 10 minutes at room temperature.

2. Add 300 l of Isopropanol and mix the contents. Keep the tube in iceor freezer for 10 minutes.

3. Spin the sample for 10 minutes at maximum speed use cold microcentrifuge or cold room for spinning samples. Decant the solution andadd ~500 l of wash solution from sides of tube and decant itimmediately.

Ethanol Wash Solution: Add all contents of bottle containing 10Xethanol wash (20 ml) into a clean bottle; add 180 ml water and 200 mlof ethanol. Prefer to keep bottle containing Ethanol Wash Solution at 4C

4. Spin the tube again for 5 minutes and remove any residual washsolution.

5. Resuspend the pellet in 50 l of sterile water or TE.

Note: Above protocol can also be used to removing contaminants fromenzymatic reactions. Protocol 2:

If you wish to remove dNTPs, follow protocol for using spin filters.

Genomic DNA Isolation (Solid Tissues)

1. Take 5-10 mg of fresh or frozen mouse tail and transfer it into amicrofuge tube containing 300 mll of Lysis Solution.

2. Add 2 l of Proteinase K Solution (25 mg/ml) and vortex the tube for3 seconds.

3. Incubate the tube at 55 to 60 degrees C overnight. Tissue maydissolve in 4 hrs if tubes are inverted periodically during incubation.

4. Add 2 l of concentrated RNAse into each tube vortex for 3 seconds.

5. Add 150 ml of Protein Precipitation Solution and vortex for 15seconds. Keep the tube in ice for 5 minutes.

6. Transfer the supernatant into a clean microfuge tube containing 400ml of 100% Isopropanol. Vortex the sample for 5 seconds.

7. Centrifuge the sample at maximum speed for 3 minutes to remove


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the precipitated protein pellet.

8. Discard the supernatant and remove traces of residual solution aftera brief spin.

9. Wash the pellet by adding 300 ml of 80% ethanol from the side of

the tube. Remove ethanol wash quickly with same pipet tip withoutdisturbing the DNA pellet. Remove any residual ethanol after a briefspin or allow the tube to dry for few minutes.

10. Add 50 ml of water or TE to dissolve the DNA pellet. You may haveto warm the tube (55 to 60 degrees C) or dissolve the DNA by pipettingthe solution up and down by using a pipet tip. Store the DNA at -20degree C for long-term storage.

composition of reagents in mol bio

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