Bacterial Media Solutions and Stocks

3 agar (200 ml)

• Add 6 grams agar to 200 ml deionized water. Autoclave to sterilize.

1.6 agar (200 ml)

• Add 3.2 grams agar to 200 ml deionized water.

• Autoclave to sterilize.

Alkaline Lysis Solution

Prepare fresh solution prior to use.

Ampicillin Stock (25 mg/ml)

• Weigh out 250 mg ampicillin. Dissolve in 10 ml ddH2O.

• Sterilize by filtering through a .22 micron filter and store at -20 degrees C.

• When working with plasmids use 100 ug/ml in the LBM + Amp medium.

• When workiing with cosmids use 200 ug/ml in the LBM + Amp medium.

  
  

Dissolve 60mg of ATP in 800ul of sterile ddH2O adjust pH to 7.0 with drops of 0.1N NaOH. Test pH using a pH paper. Adjust the volume to 1.0 ml. Filter sterilize and store at -80 degrees C.

• 10 mM ZnCl2

• 10 mM MgCl2

• 100 mM Tris pH 8.0

6.2M CsCl2

• Dissolve 42 grams of CsCl2 in 40 ml sterile 1X TE pH 8.0.

0.5 M EDTA pH 8.0 (2 liters)

• Add 336.2 g Na2EDTA to about 1400 ml deionized water.

• Add 45 g NaOH (pH should move to 8.0 as ingredients dissolve).

• Adjust volume to 2000 ml with deionized water.

40 glucose (1 liter)

• Heat 600 ml deionized water in 1 liter beaker on hot plate with stirring.

• Gradually add 400 g glucose.

• When glucose has completely dissolved pour into graduated cylinder and fill to 1000 ml with deionized water.

• Mix well and pour about 100 ml into each of several bottles.

• Autoclave to sterilize.

  
  

GTE solution

Use all sterile stock solutions. Store at 4 degrees C.

IPTG

• Dissolve 2 g of IPTG in 8 ml of dH2O in a sterile polypropylene tube.

• Adjust the volume to 10 ml with dH2O and filter through a 0.22 micron syringe filter into 1 ml aliquots and store at -20 degrees C.

LBM (1 liter)

Mix:

• 10 g Difco Bacto tryptone

• 5 g Difco Bacto yeast extract

• 5 g NaCl

• 1 ml 1M MgCl2

• Adjust volume to 1 liter with dH2O.

• Add 1.1 ml 1N NaOH. (This should bring pH to 7.2.)

• Autoclave to sterilize. Note: LB medium is prepared without MgCl2.

  
  

LBM + Amp

Prepare 1 liter LBM medium; when cool add 4 ml ampicillin stock (100 ug amp/ml media); add 8 ml if the media will be used with cosmids.

LBM plates

• Include 15 g agar with ingredients for 1 liter LBM OR: Thoroughly mix 200 ml sterile 2X LBM with 200 ml sterile melted 3 agar.

• Label and date the bottom of each plate to be poured.

• Pour approximately 30 ml LBM per plate (in a stack).

• Place a weight on the top plate to minimize to condensation and let sit overnight to solidify. Store plates in a plastic sleeve in the cold room.

10X Ligation Buffer

For plasmid subcloning:

• 0.5 M Tris pH 7.4

• 0.1 M MgCl2

• 0.1 M DTT

• 10 mM ATP

• 10 mM Spermidine

10X Ligation buffer

For Ligations in low melting temperature agarose:

-- store in 100 ul aliquots at -20 degrees C.

Lysozyme Cocktail

For large scale plasmid preps using 2 ml for each sample

• 3.0 ml 1 M Tris pH 8.0

• 9.0 ml sterile ddH2O

• 60 mg Lysozyme

• 12.0 ml - enough for 6 samples.

1 M MgCl2 (1Liter)

• Dissolve 203.31 g MgCl2*6 H2O in deionized water.

• Adjust the volume to 1 liter.

• Autoclave to sterilize.

85 100 mM MgCl2 15 glycerol

• 42.5 ml

• 100 mM CaCl2

• 7.5 ml

• 100 glycerol

• 50.0 ml total volume;

mix well and use sterile ingredients or filter sterilize

3 M Potassium Acetate

Filter sterilize. The resulting solution is 3 M potassium and 5 M acetate and has a pH of about 4.8.

5 M potassium acetate (200 ml)

• Mix 98.15 g potassium acetate with 50 ml deionized water.

• Adjust volume to 200 ml with deionized water.

PP1

• 25 sucrose

• 50 mM Tris*HCL

• pH 8.0

• 1 mM EDTA pH 8.0

PP2

• 0.1 Triton X-100

• 50 mM Tris*HCL

• pH 8.0

• 60 mM EDTA

• pH 8.0

Shelf life of 3 months

PP3

• 30 polyethelyne glycol (PEG f.wt.8000)

• 1.5 M NaCl

RNAase (10 mg/ml)

• Dissolve 100 mg RNAase A (pancreatic RNAase) in 10 ml 10mM Tris-HCl 15 mM NaCl.

• Heat to 100 degrees C (in a beaker of boiling water) for 15 minutes.

• Cool slowly to room temperature. Dispense into aliquots and store at -20 degrees C.

RNAase stock solution (5 mg/ml)

• Prepare a 10 mg/ml solution in 10mM Tris-pH7.5 15 mM NaCl.

• Heat to 100 degrees C for 15 minutes.

• Allow to cool slowly to room temperature.

• Add an equal volume of sterile 80 glycerol.

• Store at -20 degrees C.

10 SDS (100 ml)

• Add 10 g BDH brand "specially pure" sodium dodecyl sulphate to 50 ml deionized water.

• Bring volume to 100 ml with deionized water.

SOB Media

• 2 Bactotryptone

• 0.5 Yeast extract

• 10 mM NaCl

• 2.5 mM KCl

• 10 mM MgCl2

• 10 mM MgSO4

• 1.5 Agar (for plates)

SOC Media

• SOB + 20 mM Glucose

3M sodium acetate (100 ml)

• Mix 40.8 g sodium acetate with 50 ml deionized water.

• Bring volume to 100 ml with deionized water.

TCM buffer

-- filter sterilize and store in 1ml aliquots at -20 degrees C.

TE (1 liter)

• Mix: 500 ml deionized water

• 1.21 g Trizma base (final concentration of 10 mM)

• 0.34 g Na2EDTA (final concentration of 1 mM)

• Bring to 1 liter with deionized water.

• pH to 7.5 by addition of 15- 20 drops of concentrated HCl.

• Autoclave to sterilize.

Tetracycline Stock solution

• Prepare a 5 mg/ml solution in 100 EtOH.

• Store at -20 degrees C.

• Use at 10 ug/ml LB medium.

Note: Magnesium ions are antagonists of tetracycline. Do not use with medium supplemented with magnesium.

Toothpick Lysis Buffer

Adjust volume to 25 ml with dH2O. Filter sterilize with a syringe filter and store at -20 degrees C in 1 ml aliquots. Can be repeatedly thawed without harm.

X-gal

Dissolve 100 mg of X-gal in 5 ml of dimethylformamide in a sterile polypropylene tube. Aliquot 1 ml into eppendorf tubes wrapped in foil (to prevent damage by light) and store at

-20 degrees C. It is not necessary to filter sterilize X-gal solutions.