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 Cells Easy 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 Protocol PCR Purification Genomic DNA Isolation (Solid Tissues)

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

100 mg/liter

Ammonium persulfate (10%), 10 ml Dissolve 1 g ammonium persulfate (APS) in H2O and adjust the final volume to 10 ml. Always use fresh solution. Acryl-bisacrylamide mix (30%), 100 ml Dissolve 29 g acrylamide and 1 g 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

carcinogen. Use gloves and a mask when making the solution. Agar plates: Use LB media or YT media except that add 15 g BactoAgar/liter. Sterilize by autoclaving. Allow the medium to cool (do not use ice buckets) to 45C and add the appropriate amount of antibiotic. Gently mix the 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 with 0.1 N NaOH. Make final volume to 1 ml and dispense solution to small aliquots. Ammoium Acetate (10M), 100 ml Dissolve 77.1 g in 100 ml of water Acrylamide (30 %), 1 liter Dissolve 380 g of Acrylamide and 20 g of N,N-methylenebisacrylamide in total volume of 1 L Acrylamide (40 %), 1liter Dissolve 380 g of Acrylamide and 20 g of N,N-methylenebisacrylamide in total volume of 600 ml Blocking solution Make a solution of 2.5 to 5% (w/v) nonfat dried milk in TBS or PBS. Bromophenol Blue (10%), 50 ml Dissolve 5 g of Bromophenol Blue in 50 ml of sterile TE and store at room temperature. Chloramphenicol Dissolve in ethanol Make stock as 175 mg/ml ethanol and 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 room temperature. CaCl2 (1 M) Dissolve 219.08 g calcium chloride -6H2O (MW = 219.08) in H2O. Adjust final volume to Sterilize by autoclaving and store at room temperature. 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 and 450 ml H2O. Mix thoroughly and store solution at room temperature. Coomassie Blue destaining solution, I liter Mix 450 ml methanol, 100 ml acetic acid and 450 ml H2O. Mix thoroughly and store solution at room temperature. Denhardt Solution (250 x) -1 liter. Dissolve 50 g Ficoll 400, 50 g polyvinylpyrrolidone and 50 g bovine serum albumin (BSA) in 600 ml distilled H2O. Bring final volume to 1 liter with H2O. Store 25 ml aliquots at 20C.

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 500 ml. Sterilize filter and store at 4 degree C. DTT (1M), 20 ml Dissolve 3.09 g of DTT in 20 ml of sterile water. Dispense into small aliquots and store at 20 degree C. DTT (1M ), 10 ml Dissolve 1.54 g 1,4-dithio-DL-threitol (MW = 154.25) in 10 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 of a water. Incubate overnight. Autoclave the solution and make aliquots. Store DEPC treated water at room temperature. EDTA (0.5 M Na2EDTA), pH 8.0, 1 liter Dissolve 186.12 g disodium ethylenediaminetetraacetate - 2H2O (FW: 372.24) in 800 ml H2O. Add ~20 g 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 10 mg/ml in sterile water. Stir until the dye has complete dissolved. Store at 4C 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 while handling Ethidium bromide. Electrophoresis Gel Loading Buffer (100 ml) Dissolve 250 mg bromophenol 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 room temperature. Electrophoresis Gel Loading Buffer, 10 ml Glycerol 5 ml, 50X TAE 0.25 ml, 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 1 ml 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 store at 20 degree C(FW: 238.3) Kanamycin 25 mg/ml in H2O and store at 20C. (For LB media or LB plates use 25 mg/liter) LB Media: Mix 10 g Bacto-Tryptone, 5 g Bacto-Yeast extract and 10 g NaCl in 900 ml H2O. Adjust the pH to 7.0 with approximately 0.2 ml of 10 M NaOH. Bring the final volume to 1 liter with H2O Sterilize by autoclaving.

M9 minimal media: Mix 12.8 g Na2HPO4-7H2O, 3 g KH2PO4, 0.5 g NaCl and 1 g NH4Clin 900 ml H2O. Adjust the pH to 7.4 with 10 M NaOH. Adjust the 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 and 10 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) in 800 ml H2O. Adjust final volume to 1 liter and store at room temperature. 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 M Na2EDTA. Adjust pH to 7.0 with NaOH. Adjust final volume to 1 liter and store 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 room temperature. NH4OAc (10M) Dissolve 770.8 g ammonium acetate (MW = 77.08) in H2O. Adjust final volume to 1 liter. PBS (10 x), 1 liter Dissolve 80 g NaCl, 2 g KCl, 26.8 g Na2HPO4-7H2O and 2.4 g KH2PO4 in 900 ml H2O. Adjust to pH 7.4 with 1M HCl. Adjust final volume 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). Remove upper aqueous phase and add 250 ml of 0.1M Tris (pH 8.0). Remove upper aqueous phase and add 250 ml of TE buffer. Store phenil soltuon in brown bottle 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 and store 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 with 3:1 ratio. Make stock solution of loading buffer as follows: 60% glycerol, 1 mM Na2EDTA, and 0.5% Bromophenol Blue. Aliquots of stock solutions can be store at 20C.

Salmon Sperm DNA (5 mg/ml), 20 ml Cut 100 mg of Salmon Sperm DNA with scissors to small pieces and dissolve in 20 ml of water. Stir the solution by using magnetic stirrer for about 4 hours. Shear the DNA by passing through a 17-gauge needle. Dispense DNA to small aliquots and store 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 mg bromophenol blue and adjust the final volume to 100 ml with H2O. make aliquots and store them at 20C. 4 SDS PAGE (10 X), 1 liter (Running buffer) Dissolve 10 g SDS, 30.3 g Tris 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 900 ml H2O. If necessary, warm the solution to dissolve. Adjust final volume to 1 liter and store aliquots at room temperature. SOB: Mix 20 g Bacto Tryptone, 5 g Bacto yeast extract, 0.5 g NaCl and 2.5 ml 1 M KCl in 900 ml H2O. Adjust pH to 7.0 with NaOH and bring final volume to 1 liter. Sterilize the solution by autoclaving. Before using media add, add 10 ml filtered/sterile 1 M MgCl2. 4 SOC: Same as SOB media except 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 and store at room temperature. Sodium Phosphate (0.2 M) (mono-sodium), 1 liter Dissolve 27.6 g NaH2PO4 -1H2O (MW = 138) in H2O, adjust final volume to 1 liter and store at room temperature. Sodium Phosphate (0.2 M) (di-sodium), 1 liter Dissolve 53.62 g Na2HPO4-7H2O (MW = 268.1) in H2O,adjust final volume to 1 liter and store at 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: dilute the stock solution 1:10,000 in gel buffer. Sensitivity: as low as 50 pg per band dsDNA SSC (20 x), 1 liter Dissolve 175.3 g NaCl and 88.2 g sodium citrate -2H2O in 900 ml H2O. Adjust pH to 7.0 with 1M HCl. Adjust final 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 and 7.4 g Na2EDTA in 900 ml H2O. Adjust pH to 7.4 with NaOH pellets or add about 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

EDTA (pH 8.0) Final volume 1L TBE (10X), I liter 54 g Tris base 27.5 g boric acid 20 ml of 0.5 M EDTA (pH 8.0) Final volume 1L Tris-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 1L TCA, 100% (w/v) Add 1 Kg trichloroacetic acid (TCA) to 454 ml H2O to make 100% (w/v) TCA. Tris (1 M), 1 liter Dissolve 121.14 g Tris base (hydroxymethyl) amino methane (MW = 121.14) in H2O. Stir the solution with magnetic stirrer and adjust pH by adding concentrated HCl (7.4: ~ 70 ml, 7.6: ~ 60 ml, 8.0: ~ 42 ml) 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 0.5 M Na2EDTA to 900 ml H2O. Adjust final volume to 1 liter, autoclave and store at room temperature. TBE (10X), 1 liter Add 108 g Tris, 55 g Boric acid and 40 ml 0.5 M Na2EDTA in water. Adjust the final volume to 1 liter with H2O and Store at room temperature. TAE (50X), 1 liter Dissolve 242 g Tris in 800 ml H2O. Add 57.1 ml glacial acetic acid and 100 ml 0.5 M Na2EDTA. Adjust the final volume to 1 liter with H2O 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. Adjust the 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, 5.8 g Tris and 0.37 g SDS in 0.2L methanol. Add water and adjust fnal volume to 1liter. Store solution at 4C TBS (1X) , I liter (Tris Buffered Saline) Dissolve 6.05 g Tris (50 mM) and 8.76 g NaCl (150 mM) in H2O. Adjust pH to 7.4 with 1 M HCl (~ 9.5 ml) and adjust the final volume to 1 liter with distilled H2O. Store solution at 4C. TBST (1X), 1 liter (Tris Buffered Saline + Tween) Dissolve 1 ml Tween 20 in 1 liter TBS buffer. Top agar Plates: Use LB media or YT media except that add 7.5 g BactoAgar/liter. Sterilize by autoclaving. Allow the medium to cool (do not use ice buckets) to 45C and add the appropriate amount of antibiotic. Gently mix the media by swirling and pour into petri plates. Allow plates to solidify and

store at 4C. X-gal/IPTG plate Before pouring the plates, add 1 ml of 0.2M IPTG and 1 ml of X-gal (40 mg/ml) solutions. Final concentration of IPTG is 0.2 mM and X-gal is 40 g/ml X-gal (20 mg/ml), 10 ml Dissolve 200 mg 5-Bromo-4-Chloro-3-IndolylD-galactoside (X-gal) in 10 ml N,N-dimethyl formamide. Make 1 ml aliquots and store at 20C. YT Media: Mix 8 g Bacto-Tryptone, 5 g Bacto-Yeast extract and 2.5 g NaCl in 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 store at 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 a molecules.

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 room temperature. 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 paper into 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, vortex briefly 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 chemical means. Routine competent cells like HB101 or DH-5 alpha can be used for

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 minutes at 121C. Caution: certain buffers like SDS or organic solvents should not be autoclaved. To avoid bacterial contamination, some solutions may be filtered through 0.22 m filter. Making LB Media: Weight 25 g LB broth powder in a 2 L flask and add water to a final volume of 1L. Shake the contents and autoclave media for 30 minutes (liquid cycle). Make sure to add enough water (about 1L) in a tray which contains your flask containing LB media. Add appropriate antibiotic 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 2 L flask and add water to a final volume of 1L. Shake the contents and autoclave media for 30 minutes (liquid cycle). Make sure to add enough water (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 media otherwise media will solidify unevenly inside the flask. Once the temperature cools down to about 50 degree C, add appropriate antibiotic. Shake the flask thoroughly to mix the antibiotic. Pour media into plates and immediately cover plates with lids. In order to dry up any excess of moisture, leave the plates overnight at room temperature. The following day, store the plates upside down in a cold room or refrigerator. Plates are stable and can be used up to 6 months.

Standard concentration of antibiotic of agar plates:

Ampicillin Plates 75 mg/L

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 and molecular 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

4C until next day. 3. Streak culture of cells on a LB plate (without antibiotic), and incubate it overnight to get isolated colonies of E. coli cells. Alternatively, you can grow about 100 l of cells (fresh or frozen stock) in about 15 ml LB media (without antibiotic) 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 of sterile LB broth media. Note: Since E.coli cells do not have any antibiotic resistance, do not add any antibiotic. 2. Grow cells on a shaker at 37C until media growth as an OD of 0.4 to 0.5@ 600 nm you can use 1 cm pathlength cuvette to check the OD. 3. Use large capacity (250 ml) autoclaved centrifuge bottles to centrifuge cells 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 at this point. It is recommended to filter both solutions. Keep filtered solutions in ice until further use. 5. Gently re-suspend the bacteria pellet with 1/10 volume of ice-cold MgCl2 solution. If your original growth volume was 1L, add total of 100 ml of MgCl2 solution in all bottles. Do not VORTEX cells. Centrifuge the cell suspension at 4,000 RPM for 10 minutes at 4oC. Discard the supernatant. 6. Re-suspend the bacteria pellet with 1/20 volume of ice cold CaCl2 solution. If your original growth volume was 1L, add total of 50 ml of MgCl2 solution. Do not VORTEX cells. Keep this suspension on ice for at least 30 minutes. 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-cold CaCl2 solution. If your original growth volume was 1L, add 20 ml of CaCl2 solution. Add 3.5 ml of glycerol and re-suspend the competent cells. Make sure to carry all steps at or below 4C. Dispense competent cells as 100 L aliquots and freeze at -80oC.

Midi Plasmid Preparation (without kit) using silica resin:

(To buy resin in bulk, please contact primmlabs.com)

A midi-prep double-stranded DNA isolation provides 4-5 times amount of template DNA as compared to plasmid made by protocols used for making mini plasmid preparations. In this protocol, a single bacterial colony having plasmid of interest is inoculated into 15 ml of liquid media containing appropriate antibiotic. The culture is incubated further for about 16 hours. After harvesting, the cells are centrifuged and silica-based alkaline-lysis purification is performed. The approximate yield of double-stranded DNA using 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 50 ml capacity Falcon tube containing 15 ml of LB media supplemented with the appropriate antibiotic. Incubate culture with shaking at 37C for about 16 hours. 2. Harvest the cells by centrifugation at 5000 rpm for 5 minutes and decant the supernatant. The cell pellets can be frozen at -70C at this point. 3. Resuspend the cell pellets in 0.5 ml of GTE (Glucose Tris EDTA Buffer)/Lysozyme solution. Incubate the solution for 10 minutes. 4. Add 0.5 ml of Alkaline Lysis Solution. Mix gently until solution clears. Leave the solution on ice for 10 minutes. 5. Add 0.5 ml of Acetate solution. Mix gently, but avoid vortexing. Leave the solution on ice for 10 minutes. 6. Transfer the solution to a microfuge tube and centrifuge at 10,000 rpm for 20 minutes. 7. Decant the supernatant to a new microfuge tube and add 5 ul of a 20 mg/ml DNase-free RNase A. Incubate sample in a 37C water bath for 10 minutes. 8. Add 1.5 ml of silica resin and allow the DNA to bind at room temperature for 5 minutes with occasional mixing. Centrifuge the solution at 3,000 for 2 minutes. 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 washing step 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 by centrifugation at 10,000 rpm for 5 minutes.

12. Transfer the supernatant into a clean 1.5 ml microfuge tube. If necessary, further purification could be carried out by ethanol precipitation Procedure 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 10,000 rmp. Wash the pellet with 70 ethanol, Resuspend the final plasmid DNA in 300 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 mL Final Volume: 200 mL Autoclave and store solution at 4C GTE/Lysozyme buffer: GTE Buffer: 38.0 mL Lysozyme Cocktail: 2.0 ml

Lysozyme Cocktail: Tris (1 M pH 8.0): Sterile H2O: Lysozyme: Final Volume: Lysis Solution: NaOH ((10M): SDS (10%): Final Volume: 8 mL

3.0 ml 60 mg 9.0 ml 12 ml

80 mL 800 mL

Acetate Solution: Pottasium acetate: 245.5 Grams Glacial acetic acid: 57 mL Final Volume: 500 mL ******

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

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

mini plasmid preparations. In this protocol, a single bacterial colony having plasmid of interest is inoculated into 15 ml of liquid media containing appropriate antibiotic. The culture is incubated further for about 16 hours. After harvesting, the cells are centrifuged and phenol/chloroform based purification is performed. The approximate yield of double-stranded DNA using 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 50 ml capacity Falcon tube containing 15 ml of LB media supplemented with the appropriate antibiotic. Incubate culture with shaking at 37C for about 16 hours. 2. Harvest the cells by centrifugation at 5000 rpm for 5 minutes and decant the supernatant. The cell pellets can be frozen at -70C at this point. 3. Resuspend the cell pellets in 0.5 ml of GTE (Glucose Tris EDTA Buffer)/Lysozyme solution. Incubate the solution for 10 minutes. 4. Add 0.5 ml of Alkaline Lysis Solution. Mix gently until solution clears. Leave the solution on ice for 10 minutes. 5. Add 0.5 ml of Acetate solution. Mix gently, but avoid vortexing. Leave the solution on ice for 10 minutes. 6. Transfer the solution to a microfuge tube and centrifuge at 10,000 rpm for 20 minutes. 7. Decant the supernatant to a new microfuge tube and add 5 ul of a 20 mg/ml DNase-free RNase A. Incubate sample in a 37C water bath for 10 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 top aqueous 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 top aqueous phase and transfer into a fresh microfuge tube. Add 1/10 volume of 3M sodium acetate (pH 7.0) and 3 volumes of ethanol.

11. Spin sample for 10 minutes at 10,000 rmp. Remove supernatant and pulse in the microfuge and remove the remainder of the liquid. Resuspend the final plasmid DNA in 300 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 mL Final Volume: 200 mL Autoclave and store solution at 4C GTE/Lysozyme buffer: GTE Buffer: 38.0 mL Lysozyme Cocktail: 2.0 ml Lysozyme Cocktail: Tris (1 M pH 8.0): Sterile H2O: Lysozyme: Final Volume: Lysis Solution: NaOH ((10M): SDS (10%): Final Volume: Acetate Solution: Pottasium acetate: Glacial acetic acid: Final Volume: ******

3.0 ml 9.0 ml 60 mg 12 ml 8 mL 80 mL 800 mL 245.5 Grams 57 mL 500 mL

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

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

acetate. Following lysis, solution is centrifuged and phenol/chloroform based purification is performed. The collected DNA is concentrated by ethanol precipitation. The approximate yield of double-stranded DNA using this method is approximately 1 g of DNA per ml of cell culture. 1. Transfer 1.5 ml of overnight bacterial culture into a separate microfuge tube and centrifuge for 2 minutes at 10,000 rpm. Remove the clear supernatant. Add a further 1.5 ml of bacterial culture to the tube. Spin and remove the supernatant as before. Pellet represents 1.5 ml of culture. 2. Resuspend the cell pellets in 0.2 ml of GTE (Glucose Tris EDTA Buffer)/Lysozyme solution. Incubate the solution for 10 minutes. 3. Add 0.2 ml of Alkaline Lysis Solution. Mix gently until solution clears. Leave the solution on ice for 10 minutes. 4. Add 0.2 ml of Acetate solution. Mix gently, but avoid vortexing. Leave the solution on ice for 10 minutes. 5. Transfer the solution to a microfuge tube and centrifuge at 10,000 rpm for 10 minutes. 6. Decant the supernatant to a new microfuge tube and add 2 ul of a 20 mg/ml DNase-free RNase A. Incubate sample in a 37C water bath for 5 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 upper aqueous layer to a fresh tube. There will be two distinct layers with an interface that may have white denatured proteins. Carefully remove the upper aqueous layer without disturbing the denatured protein or phenol layer. 9 . Spin the tube for 5 minutes at 10,000 rpm. Gently remove the top aqueous 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 top aqueous phase and transfer into a fresh microfuge tube. Add 1/10 volume of 3M sodium acetate (pH 7.0) and 3 volumes of ethanol. Place the tube at 20oC or at -70oC for 20 minutes.

11. Spin sample for 10 minutes at 10,000 rmp. Remove supernatant and pulse in the microfuge and remove the remainder of the liquid. Resuspend the 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 are enough for about 75 midi preps. Midi preps for following protocol will take almost same time as mini preps. Also, there is no significant cost difference in growth media for midi verses mini preps. Therefore, it is recommended to use this kit for making midi preps only-the yield will be sufficient for the screening of positive colonies and for subsequent molecular biology applications. This protocol is designed to make midi plasmid preparations which could give as high as ~75 g of high copy plasmid DNA. You can scale down the procedure for mini preps. CONTENTS: Resuspension Solution: Lysis Solution: Neutralization Solution: Precipitation Solution: Ethanol wash: Complimentary Solutions: 1X TE (Tris EDTA Buffer) 10 X TE (Tris EDTA Buffer) Gel Running Buffer (50 X TAE) 30 ml 30 ml 50 ml 30 ml 30 ml 30 ml 100 ml (in brown bottles) 20 ml (10X)

(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 growth of 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 1 minute to remove residual media. 2. Re-suspend the pellet in 300 l ml of Re-suspension Solution. Vortex to

re-suspend the pellet. The bacterial pellet should be re-suspended completely until no cell clumps are visible. Let the cell suspension stand at room temperature for 5 minutes. 3. Add 300 l of Lysis Solution and mix the contents gently until the cell suspension 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 brief vortexing. Let the tube stand for 5 minutes at room temperature. Transfer the 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 speed for 5 to 15 minutes. 6. In the mean time, dispense 1.2 ml of DNA Precipitation Solution into 2 ml of provided microfuge tubes. 7. Add 600 l of clear supernatant (step 5) into tube containing DNA Precipitation solution. Vortex the solution and keep tubes in ice or at 20C for 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 (~1 ml) discard it immediately. In order to avoid dislodging the pellet, it is recommended to add ethanol wash from sides of the tube. Note: DNA pellet may 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 wash into a clean bottle; add 180 ml water and 200 ml of ethanol. Keep bottle containing working Ethanol Wash Solution at 4C. Wash solution is stable for 1 year. 10. Spin tubes for 5 minutes and remove any traces of ethanol wash with a pipette tip. 11. Add 100 l of water or TE. Keep tubes at 37 to 42 degree C water bath for about 10 minutes. Vortex tubes briefly to dissolve the DNA.

Mini Preps:
Scale down the solutions according to growth media. Protocol is same as above except minor changes as below: 1. Avoid washing step (step # 9): after spinning the tube (step # 8), discard solution and spin the tube again for about 5 minutes in cold room or use cold centrifuge. 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

tubes briefly to dissolve the DNA. -----------------------------------

Easy Pure Maxi/Mega plasmid preparation kit

(20 Preparations) Following protocol is designed for making maxi plasmid preparations, which can be used for most of molecular biology applications such as transfection experiments, cloning, restriction digestion or PCR. This kit provides reagents for 20 maxi plasmid preps. Important features of our Maxi plasmid isolation kit are: * Extremely simple to perform. * You get up to 5 mg of DNA/-0.5L culture or ~95% of the amount present in the 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: Lysis Solution: Neutralization Solution: Suspension Solution: Precipitation Solution A: Precipitation Solution B: Concentrated RNAse: 1X TE (Tris EDTA Buffer): 10 X TE (Tris EDTA Buffer): 200 ml 200 ml 200 ml 20 ml 40 ml 20 ml 250 l 30 ml 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 vigorous shaking. Pellet the cells by centrifugation at 5000 rpm for 10 minutes. Resuspend the pellet in 10 ml of Re-suspension Solution. The pellet should be

re-suspended thoroughly until no cell clumps are visible. Let the cell suspension stand at room temperature for 5 minutes. Note: Growth of 500 ml culture of high copy plasmid may contain as high as 5 mg of plasmid DNA. Unlike column or resin based procedures, this procedure is designed to isolate entire amount of plasmid DNA from your culture. Therefore, depending upon your needs, you may scale up or down your preparations. Suggestion: If a large amount of DNA prep is required but you do not wish to process at the same time, follow following simple steps: Grow media in 1-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 not affect the quality of DNA. Take 2 ml of re-suspended pellet and add 8 ml of Re-suspension solution. The rest of procedure is same. 2. Add 10 ml of Lysis Solution and mix the contents gently by inverting the tube 5-6 times. Let the solution stand for 5 minutes at RT. Do not use vortex to mix the contents. 3. Add 10 ml of Neutralization Solution and mix the contents by brief vortexing (5 seconds). Keep the solution in ice for about 10 minutes. Spin the 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 tube by using a cheese cloth. Note: If solution is not fully transparent, some precipitates may have gone into 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 at 12, 000 rpm. The temperature during spinning could be set anywhere from 4 to 15C. Discard the supernatant completely and spin the tube again (2-5 minutes) 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 10 minutes at room temperature. If the solution is not clear, spin the tube for 1 minute at maximum speed to remove any insoluble impurities. Transfer the clear 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

brief vortexing and incubates tubes at room temperature for at least 10 minutes. Add 0.4 ml of Precipitation Solution B in each tube and vortex tube to mix the contents. Centrifuge tubes in a cold centrifuge for 10 min at maximum speed to precipitate the DNA. Note: After incubation, it is safe keep the tubes in refrigerator (~4C) until next day. 7. Remove the supernatant; rinse the pellet gently with 80% ethanol. Make sure that do not loose the pellet. Spin the tubes briefly for few seconds and carefully remove traces of ethanol wash without losing the DNA pellet. If you need 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 point the DNA pellet may be quite compact but place tubes containing DNA in a water bath (~42C) for 10 minutes. Vortex or pipet the solution up and down to 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 tube tubes rigorously for 30 seconds. Keep tube in ice for 5 minutes. Pellet the DNA by spinning tubes in a cold centrifuge for 10 min at maximum speed. Carefully remove ethanol wash. Note: Prefer to transfer the ethanol wash into a new tube to avoid accidentally throwing away the DNA pellet. Note: Take the spectrophotometric reading of DNA in a TE buffer. Avoid taking the reading in water as it can cause a shift in the OD due to an acidic pH of the water.

Large Scale Cesium Chloride Plasmid Preparation (without kit):

The method used for the preparation of large-scale plasmid DNA by using an alkaline lysis followed by ultracentrifugation. in cesium chloride-ethidium bromide gradients. Briefly, grown culture containing the desired plasmid is centrifuged, pellet is incubated in a lysozyme buffer, and treated with alkaline lysis reagents. Lysis buffer is used to solubilize proteins and membranes are precipitated with low pH acetate buffer. After centrifugation, the lysate is cleared by filtration through cheesecloth. The Plasmid-containing supernatant is transferred to a new tube, and the plasmid DNA is precipitated and the DNA pellet is resuspended in a buffer containing cesium chloride and ethidium bromide. DNA solution is loaded into special plastic tubes and subjected to ultracentrifugation for about 16 hours. High speed centrifugation establishes cesium chloride density gradient, and the ethidium 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

isobutanol. The final DNA pellet is resuspended in TE buffer and assayed by agarose gel electrophoresis. 1. Inoculate 10 ml LB (+ Antibiotic) with a single colony or 10 L of frozen glycerol stock and grow culture for about 16 hours or overnight at 37C. 2. Transfer 5 ml of the culture to 1 L of similar media and incubate for an additional 8-10 hours or overnight with vigorous shaking at 37C. 3. Transfer the cells to 500 mL centrifuge bottles and spin at 5000 RPM for 20 minutes. 4. Decant the supernatant, save the cell pellet. The cell pellets can be frozen at -70 C at this point. 5. Resuspend the cell pellet in 40 mL GTE/Lysozyme buffer. Gently break the pellet with a pipette and incubate for solution for10 minutes at room temperature. Do not vortex the lysate as it may shear the chromosomal DNA. 6. Add 40 ml of Alkaline Lysis Solution. Mix gently until solution clears. 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 at 10,000 rpm for 30 minutes at 4C by using the GSA or equivalent rotor. 9. Filter the supernatant through cheesecloth into fresh centrifuge bottles. 10.Add 90 ml of HPLC grade Isopropanol. Swirl to mix, and incubate in an 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 combined total of 32 ml TE buffer, 5 ml of 5 mg/ml ethidium bromide, and 37 g cesium chloride. Final concentration of cesium chloride should be 1 g/ml). 13.Transfer the sample into 35 ml plastic ultra- centrifuge tubes, remove air bubbles, seal with rubber stoppers, and crimp properly. Note: Use TE to bring volume to just below the neck. Quick-Seal tubes (from Beckman cat # 342412) can also be used. 14.Centrifuge overnight (16-20 hours) at 60,000 rpm at 15-20C in the Sorvall OTD-75B ultracentrifuge (DuPont) using the T-865 or NVT90 or VTi80 rotor. 15.Carefully remove the centrifuge tubes from the rotor. And Place in clamp stand. Visualize the ethidium bromide stained DNA band under long-wave UV light, and remove the lower DNA band using a 5 or 10 cc syringe. Sometimes, two pink bands are too close to each other and it may be helpful first to remove and discard the top band which is chromosomal DNA. 16.Transfer the plasmid DNA from syringe to 15 mL Falcon tube. Add 10 mL of NaCl/water saturated n-butanol. Vortex the mixture and spin in

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: Ethidium bromide is carcinogenic. 18.Repeat butanol extraction 3 to 5 times until the organic phase becomes clear. 19.Transfer clear DNA solution to a clean JA17 centrifuge tube. Add 1/10th volume 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 room temperature. 21.Discard supernatant and resuspend pellet in 2 ml of 0.3 M Na Ac pH 5.2. Add 6 mL of 100% ethanol and spin DNA at 10,000 RPM for 20 minutes. 22.Wash the pellet carefully with 70% ethanol. Dry the pellet using Speed-Vac and Resuspend the pellet in 500 l of TE by vortexing. 23.Store plasmid DNA in freezer. Buffers: GTE (Glucose Tris EDTA Buffer): Glucose: 1.8 Grams (1M) Tris pH 8.0: 5.0 mL (0.5M) EDTA: 4.0 mL Final Volume: 200 mL Autoclave and store solution at 4C GTE/Lysozyme buffer: GTE Buffer: 38.0 mL Lysozyme Cocktail: 2.0 ml Lysozyme Cocktail: Tris (1 M pH 8.0): 3.0 ml Sterile H2O: 9.0 ml Lysozyme: 60 mg Final Volume: 12 ml 12 ml - enough for 6 samples, prepare 2 ml aliquots (fresh each time). Lysis Solution: NaOH ((10M): SDS (10%): Final Volume: 8 mL

80 mL 800 mL

Acetate Solution: Pottasium acetate: 245.5 Grams Glacial acetic acid: 57 mL Final Volume: 500 mL

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

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 while retaining small contaminants such as salts and small nucleotides.

Protocol #1

Protocol for the extraction of DNA fragments from high melt agarose gels. (This protocol does not require gel filtration matrix)

1. Excise the gel slice from a high melt agarose containing the DNA band of interest. Slice gel into small pieces and place it inside the spin filter. Insert the spin filter into receiving tube. This protocol is suitable for high melt agarose gel only, do not use low melt agarose. We also recommend using 0.5 X TAE buffer for agarose gel electrophoresis. Optional step: In order to increase the recovery of DNA, place the spin filter (containing your gel slice) at -80 degree centigrade for at least 10 minutes. However, you can keep it at 80 degree centigrade for indefinite 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 directly for subsequent reactions such as labeling, DNA ligation, PCR, fillin/kinase reaction with the Klenow fragment or four deoxynucleotides and rATP sequencing reactions etc.,

Note: Recovery of DNA is usually between 60 to 80% - depends upon size and concentration of DNA. You may verify the DNA concentration by using small aliquot of eluted DNA and run it on a gel. If you are planning to use eluted DNA for DNA ligation, it is recommended that you run the gel electrophoresis by using 0.5X TAE as running buffer. After extracting the DNA from gel slice, add an equal amount of water to the eluted DNA from the spin filter. Salt concentration will be low enough and will not interfere with subsequent ligation reactions. If you wish to remove TAE buffer completely or to concentrate DNA, follow protocol # 2. Do not directly concentrate eluted DNA (from agarose gel slices) with ethanol as any trace of agarose will coprecipitate with your DNA which will make it difficult to resuspend your precipitated DNA.

Protocol #2

Purification 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 cycle sequencing 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 swelled in about 5 hours or leave overnight at room temperature. Once Sephadex is fully swelled, store it at 4 degree centigrade for further use. It is recommended that you make few aliquots of hydrated gel in separate tubes for further use. Hydrated gel with sufficient amount of sterile 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 Sephadex containing tube. This should leave you with thick slurry of Sephadex G50. Add ~700 l of Sephadex G-50 slurry into a spin column. Insert spin 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 with additional Sephadex G-50 slurry and spin once again for two minutes @ 2000 RPM. 3. Insert spin filter back into receiving tube and transfer 20 to 50l of the sample to the center of gel matrix without disturbing the Sephadex bed surface. Centrifuge for 3 minutes @ 3000 RPM. Store eluted DNA at -20 degree centigrade for subsequent uses. Note: Care must be taken to place the sample directly in the middle of the Sephadex gel surface of the column. Touching the sides of the column may reduce the separation efficiency. Load the sample within 10 minutes to avoid drying of the gel.

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

Standard Conditions for PCR Protocol PCR is highly sensitive to contamination which can cause the amplification 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 a cocktail and reagent or sample. Most of the companies that provide thermostable enzyme also supply buffer for PCR reactions. Optimum PCR product depends upon template 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 10x PCR Buffer dNTP (25 mM each dNTPs) Primer 1 (add 40 p mol) Primer 2 (add 40 p mol)

85 10 1 0.4 0.4

l l l l (from stock of 100 pmol/l) l (from stock of 100 pmol/l)

DNA Template Taq or Vent Pol

1 2

l (~100 to 500 g) 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 at 20 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 1 2 3 4 5 6 7 Temp C 95 (Denaturation) 95 (Denaturation) 58 (Annealing) 72 (Extension) 30 cycles steps 2 to 4. 72 (Extension) END 15.00 1 Time (minutes) 5:00 1.00 1.00 1.:00 cycles 1 (see step 5) (see step 5) (see step 5)

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 Purification
Protocol 1: Application: This protocol purifies and removes most
of contaminants from PCR product. This protocol will also denatures thermostable enzyme which will not interfere during subsequent reactions such as sequencing or restriction enzyme digestion of PCR products.

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

1. Transfer 100 l of PCR reaction mixture into a new microfuge tube. Add 300 l of PCR-DNA Purifier/Precipitation Solution. Vortex the mixture 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 ice or freezer for 10 minutes. 3. Spin the sample for 10 minutes at maximum speed use cold micro centrifuge or cold room for spinning samples. Decant the solution and add ~500 l of wash solution from sides of tube and decant it immediately. Ethanol Wash Solution: Add all contents of bottle containing 10X ethanol wash (20 ml) into a clean bottle; add 180 ml water and 200 ml of ethanol. Prefer to keep bottle containing Ethanol Wash Solution at 4C 4. Spin the tube again for 5 minutes and remove any residual wash solution. 5. Resuspend the pellet in 50 l of sterile water or TE. Note: Above protocol can also be used to removing contaminants from enzymatic 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 a microfuge tube containing 300 mll of Lysis Solution. 2. Add 2 l of Proteinase K Solution (25 mg/ml) and vortex the tube for 3 seconds. 3. Incubate the tube at 55 to 60 degrees C overnight. Tissue may dissolve 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 15 seconds. Keep the tube in ice for 5 minutes. 6. Transfer the supernatant into a clean microfuge tube containing 400 ml of 100% Isopropanol. Vortex the sample for 5 seconds. 7. Centrifuge the sample at maximum speed for 3 minutes to remove

the precipitated protein pellet. 8. Discard the supernatant and remove traces of residual solution after a 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 without disturbing the DNA pellet. Remove any residual ethanol after a brief spin or allow the tube to dry for few minutes. 10. Add 50 ml of water or TE to dissolve the DNA pellet. You may have to warm the tube (55 to 60 degrees C) or dissolve the DNA by pipetting the solution up and down by using a pipet tip. Store the DNA at -20 degree C for long-term storage.