Common Solutions For Molecular Biology Techniques. MOLARITY CALCULATOR
	Molar/Molarity
	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
	Easy Pure Maxi/Mega Plasmid Preparation Kit
	Easy clean DNA Spin Filters
	PCR Purification
	Genomic DNA Isolation (Solid Tissues)

Common Solutions For Molecular Biology Techniques

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 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 4°C. 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 Bacto-Agar/liter. Sterilize by autoclaving. Allow the medium to cool (do not use ice buckets) to 45°C 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 4°C.

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

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 – 20°C.
(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 –20°C.

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 –20°C.

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 4°C 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 ß-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 µm filtration and store at – 20°C.

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 – 20°C.
(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 µm filter. Store at 4°C

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 4°C.

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 4°C.

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 – 20°C.

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 – 20°C. 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 - 20°C. 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 µl 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 4°C

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 4°C.

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 Bacto-Agar/liter. Sterilize by autoclaving. Allow the medium to cool (do not use ice buckets) to 45°C 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 4°C.

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 µg/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 aliquots and store at – 20°C.

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 (Avogadro’s number) parts of a molecules.

Autoclave- Sterilization time:
Usually 15 minutes to 30 minutes at 121°C. Caution: certain buffers like SDS or organic solvents should not be autoclaved. To avoid bacterial contamination, some solutions may be filtered through 0.22 µm 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 µg/ml of ds DNA
I OD (at 260 nm) = 33 µg/ml of ss DNA
I OD (at 260 nm) = 40 µg/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

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 µg/ml of ds DNA
I OD (at 260 nm)= 33 µg/ml of ss DNA
I OD (at 260 nm) = 40 µg/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 4°C 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 µl of cells (fresh or frozen stock) in about 15 ml LB media (without antibiotic) – and incubate overnight at 37°C 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 37°C 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 4°C. 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 4°C. Dispense competent cells as 100 µL aliquots and freeze at -80oC.

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

2. Add 300 µl 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 µl 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 µl of sterile water or TE.

Note: Above protocol can also be used to removing contaminants from enzymatic reactions.

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Protocol 2:

If you wish to remove dNTP’s, follow protocol for using spin filters.

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 µg of high copy plasmid DNA. You can scale down 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 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 µl 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 µl 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 µl 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 µl 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 µl 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 µl 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.

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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 200 ml
Lysis Solution 200 ml
Neutralization Solution 200 ml
Suspension Solution 20 ml
Precipitation Solution 40 ml
Concentrated RNAse 250 µl
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 vigorous shaking. 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 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.

DNA Purification

6. Add 1 ml of DNA Precipitation Solution in each tube. Mix the contents by brief vortexing and incubates tubes at room temperature for at least 10 minutes. Centrifuge the tube 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 µl 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.

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