Alkaline Lysis Buffer A Recipe

1. Add the following to 900ml distilled H2O 9g Glucose 3g Tris 20ml of 0.25M EDTA

2. q.s. to 1000ml with distilled H2O3. Autoclave using standard conditions4. Allow to cool to room temperature5. Store at 4oC

When ready to use add 0.02g lysozyme to 100ml of alkaline lysis buffer A to yield alkaline lysis buffer 1.

Alkaline Lysis Buffer 2 Recipe

1. Add the following to 100ml distilled H2O 5ml of 20% SDS 2ml of 10M NaOH

Alkaline Lysis Buffer 3 Recipe

1. Add the following to 100ml distilled H2O 29.4g KAc 11.5ml of Glacial Acetic Acid

Carmalum Stain Recipe

Stock solution

1. Add 10g of carmalum to 200ml of distilled water2. Slowly add in 5ml of glacial acetic acid3. Boil for approximately 1 hour4. Allow to cool5. Filter

Carmalum stain is used for cell nuclei.

Cautions: ALWAYS wear personal protective equipment when in the laboratory with concentrated acids (or any other time)! Always add acid slowly into water and do not add water into acid!

Coomassie Blue Solution Recipe

1. Dissolve 2g Coomassie Blue (Serva Blau) in 250ml water2. Slowly add 75ml of glacial acetic acid3. Add 500ml of ethanol4. q.s. to 1000ml with water

Final concentrations:0.2% Coomassie Blue7.5% Acetic Acid50% Ethanol

LB Agar Recipe

1. Add the following to 800ml H2O 10g Bacto-tryptone 5g yeast extract 10g NaCl

2. Adjust pH to 7.5 with NaOH3. Add 15g agar4. Melt agar into solution in the microwave5. Adjust volume to 1L with dH2O6. Sterilize by autoclaving

20X SSC Recipe

1. Dissolve the following in 800ml of distilled H2O. 175.3g of NaCl 88.2g of sodium citrate

2. Adjust the pH to 7.0 with a few drops of 1M HCl.3. Adjust the volume to 1L with additional distilled H2O.4. Sterilize by autoclaving.

10X Xilene Cyanol/Bromophenol Blue DNA loading buffer Recipe

1. Dissolve in 6.25 ml of H2Oo .025g of Xilene cyanolo .025g of Bromophenol Blueo 1.25ml of 10% SDSo 12.5ml of glycerol

Note: Sucrose (40%), Ficoll (15%), or glycerol (30%) can be used in place of each other in DNA loading buffer

50X TAE Recipe

1. Add the following to 900ml distilled H2O 242g Tris base 57.1ml Glacial Acetic Acid 18.6 g EDTA

2. Adjust volume to 1L with additional distilled H2O

10X TBE RecipeAdd the following to 800ml H2O

108g Tris base. 55g Boric acid 9.3g EDTA

2. Adjust volume to 1L with additional distilled dH2O

1X TE Recipe

3.[1.] Add the following to 990ml distilled H2O 10ml  1M Tris-HCl (pH 8.0) 400µl  0.25 M EDTA

5X Tris Glycine Buffer Recipe

1. Dissolve in 700 ml of H2O: 15.1g Tris base 94g glycine 50ml of 10% SDS

2. After solid is dissolved, adjust volume to 1L with H2O

X-gal staining solution Recipe

To 495ml of PBS add

1. 1.050g Ferrocyanide2. 0.825g Ferricyanide3. 0.050g NaDeoxycholate4. 0.2g MgCl2

5. 500ul Nonidet-P406. 0.5g X-gal in 5ml N,N-Dimethylformamide

Protocol for E. coli Competent Cell Preparation

1. Inoculate 5ml of L-broth with a single colony of selected E. coli strain and incubate overnight at 37o C. with moderate agitation(~250rpm)

2. Inoculate 50ml of L-broth with ~100-300ml of the overnight culture and incubate at 37o C. with moderate agitiation(~250rpm) until the A595=0.375 ( initial inoculum should have A595<0.1)

3. Place culture on ice in autoclaved, prechilled Corex tubes for 10 minutes4. Centrifuge cells 7 minutes at 2500rpm(+4o C) and then resuspend pellet (very gently by

hand-do not vortex) in 10ml CaCl2 solution( 60mM CaCl2, 15% glycerol, 10mM PIPES, pH 7.0)

5. Centrifuge cells 5 minutes at 2500rpm(+4o C) and then resuspend pellet (very gently by hand-do not vortex) in 10ml CaCl2 solution

6. Leave cells on ice for 30 minutes and then centrifuge for 5 minutes at 2500rpm(+4o C) and resuspend cells (very gently by hand-do not vortex)in 2ml CaCl2 solution

7. Aliquot final preparation in 100ml aliquots on dry ice and store at -70o C. (cells should be viable for up to a year at -70o C)

L-broth: 1% bactotryptone, 1% NaCl, 0.5% yeast extract

Phenol Purification of DNA From Low Melting Point Agarose

1. Place phenol mix at 37oC for at least 1 hour2. Melt excised agarose fragment at 65oC. for 10 minutes or until agarose is molten, then

place molten agarose at 37oC for 2 minutes3. Add 0.5X volume of phenol mix and shake vigorously for 45 seconds4. Centrifuge for 5 minutes at room temperature and transfer aqueous layer to clean tube5. Repeat phenol extraction and centrifugation step6. Transfer aqueous layer to a clean tube and add 1/50 volume of 5M NaCl, shake for 45

seconds, and centrifuge for 5 minutes at room temperature7. Transfer aqueous material to a clean tube, add 2X volume of EtOH and precipitate (either

15 minutes at -70oC. or at least 1 hour at -20oC)8. Centrifuge for 15 minutes and aspirate off aqueous material9. Wash pellet in 1X volume of 70% EtOH and repeat centrifugation10. Aspirate off aqueous material and air dry pellet for 5 minutes (or dry in speedvac for ~2

minutes)11. Resuspend pellet in 20µl TE buffer

Phenol mix: 25ml pure phenol, 15ml H2O, 400ml 1M Tris-HCl, pH to 8.0 with 2 drops 5M NaCl

The usual stock solution concentration of EtBr is 10 mg/ml, or 1000x higher. Are you sure you need the lower concentration?I would make this by first making a 10 mg/ml stock and diluting 1000x (assuming that this is what you really need).

-phage434-

By default,EtBr is actually 10mg per mlwe used to convert it to .5 microgram per ml

-Mazhar Hussain-