Bacterial Media Solutions and Stocks

 

3 agar (200 ml)

 

 

1.6 agar (200 ml)

 

Alkaline Lysis Solution

 

stock solution	volume	final concentration
1N NaOH	2.0 ml	0.2 N
10 SDS	1.0 ml	1
sterile ddH2O	7.0 ml
	10.0 ml

Prepare fresh solution prior to use.

 

Ampicillin Stock (25 mg/ml)

 

 

• Add 6 grams agar to 200 ml deionized water. Autoclave to sterilize.
  • Add 3.2 grams agar to 200 ml deionized water.
  • Autoclave to sterilize.
  • 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.

     

     

100mM ATP

 

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.

 

6.2M CsCl2

 

 

0.5 M EDTA pH 8.0 (2 liters)

 

40 glucose (1 liter)

 

GTE solution

 

stock solution	volume	final concentration
40 sterile glucose	2.27 ml	50 mM
0.5 M EDTA pH 8	2.0 ml	10 mM
1M Tris-HCl pH 8	2.5 ml	25 mM
sterile ddH2O	93.23 ml
	100.0 ml

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

IPTG

LBM (1 liter)

Mix:

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

 

10X Ligation Buffer

For plasmid subcloning:

 

10X Ligation buffer

For Ligations in low melting temperature agarose:

 

Stock solution	Volume	Final Concentration
1M Tris.HCl pH7.5	660 uli	0.66M
1M MgCl2	50 ul	50 mM
1M DTT	50 ul	50 mM
100 mM ATP	100 ul	10 mM
sterile ddH2O	140 ul
	1000 ul

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

 

Lysozyme Cocktail

For large scale plasmid preps using 2 ml for each sample

 

 

1 M MgCl2 (1Liter)

 

85 100 mM MgCl2 15 glycerol

3 M Potassium Acetate

 

stock solution	volume
5 M KOAc	60 ml
glacial acetic acid	11.5 ml
ddH2O	28.5 ml
	100 ml

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)

 

PP1

 

PP2

 

Shelf life of 3 months

PP3

 

RNAase (10 mg/ml)

 

RNAase stock solution (5 mg/ml)

 

10 SDS (100 ml)

 

SOB Media

 

SOC Media

 

3M sodium acetate (100 ml)

 

TCM buffer

 

		final concentration
1M Tris.HCl pH7.5	100 ul	10 mM
1M CaCl2	100 ul	10 mM
1M MgCl2	100 ul	10 mM
sterile ddH2O	9.7 ml
	10.0 ml

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

 

TE (1 liter)

 

 

Tetracycline Stock solution

 

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

 

Toothpick Lysis Buffer

 

1.25 ml	1N NaOH
0.25 ml	0.5M EDTA pH 8.0
0.625 ml	10 SDS
1.75 g	Ficoll
250 ul	1 Bromophenol blue dye

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.

10X CIP Buffer
• • 10 mM ZnCl2
  • 10 mM MgCl2
  • 100 mM Tris pH 8.0
  Dissolve 42 grams of CsCl2 in 40 ml sterile 1X TE pH 8.0.
  • 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.
  • 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.

     

  • 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.
  • 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.

     

  • 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.
  • 0.5 M Tris pH 7.4
  • 0.1 M MgCl2
  • 0.1 M DTT
  • 10 mM ATP
  • 10 mM Spermidine
  • 3.0 ml 1 M Tris pH 8.0
  • 9.0 ml sterile ddH2O
  • 60 mg Lysozyme
  • 12.0 ml - enough for 6 samples.
  • Dissolve 203.31 g MgClundefined6 H2O in deionized water.
  • Adjust the volume to 1 liter.
  • Autoclave to sterilize.
  • 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
• • Mix 98.15 g potassium acetate with 50 ml deionized water.
  • Adjust volume to 200 ml with deionized water.
  • 25 sucrose
  • 50 mM TriundefinedHCL
  • pH 8.0
  • 1 mM EDTA pH 8.0
  • 0.1 Triton X-100
  • 50 mM TriundefinedHCL
  • pH 8.0
  • 60 mM EDTA
  • pH 8.0
  • 30 polyethelyne glycol (PEG f.wt.8000)
  • 1.5 M NaCl
  • 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.
  • 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.
  • Add 10 g BDH brand "specially pure" sodium dodecyl sulphate to 50 ml deionized water.
  • Bring volume to 100 ml with deionized water.
  • 2 Bactotryptone
  • 0.5 Yeast extract
  • 10 mM NaCl
  • 2.5 mM KCl
  • 10 mM MgCl2
  • 10 mM MgSO4
  • 1.5 Agar (for plates)
  • SOB + 20 mM Glucose
  • Mix 40.8 g sodium acetate with 50 ml deionized water.
  • Bring volume to 100 ml with deionized water.
  • 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.
  • Prepare a 5 mg/ml solution in 100 EtOH.
  • Store at -20 degrees C.
  • Use at 10 ug/ml LB medium.