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Lambda Methods Media and Solutions Updated Nov 29 1990
C. Helms
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.
75 mM calcium chloride (100 ml):
- Mix 830 mg calcium chloride with 100 ml deionized water.
- Autoclave to sterilize.
DEAE-cellulose
DE52 (Whatman preswollen) is prepared in the hydrogenated form as follows: - In a 2L flask suspend 500 g of DE52 in 1.5L of 0.1N HCl. Once the DE52 has settled decant the check the pH. If the pH > 2.0 repeat the above step with fresh 0.1N HCl. If the pH <= 2.0 rinse DE52 with 0.1M Tris-HCl pH8.0 until the pH of the decanted supernatent is > 7.0. Follow this with one rinse of dH2O and 3 rinses of 10mM Tris-HCl pH8. Store DE52 at 4deg.C as 1:1 slurry in 10 mM Tris-HCl pH8. Before use mix the slurry well and bring to room temperature.
DNaseI stock solution
- Prepare a 1 mg/ml solution in 40% glycerol containing 150mM NaCl.
- Store at -20deg.C
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.
4 mM ferric chloride (100 ml)
- Mix 110 mg ferric chloride with 100 ml deionized water.
- Autoclave to sterilize.
2% gelatin (200 ml)- Mix 4 grams gelatin with 200 ml deionized water.
- Autoclave to sterilize.
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.
Lambda diluent (100 ml)- Mix: 1 ml sterile 1M Tris-HCl pH8
- 0.2 ml sterile 1M MgCl2
- 98.8 ml sterile dH2O
Lambda diluent + gelatin(100 ml)- Mix: 1.0 ml sterile 1M Tris-HCl pH8
- 0.2 ml sterile 1M MgCl2
- 0.5 ml 2% gelatin
- 98.3 ml sterile dH2O
LB (1mM MgCl2 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.
2X LB (1mM MgCl2 1 liter) - Mix: 20 g Difco Bacto tryptone
- 10 g Difco Bacto yeast extract
- 10 g NaCl
- 2 ml 1M MgCl2
- Adjust volume to 1 liter with dH2O.
- Add 2.2 ml 1N NaOH. (This should bring pH to 7.2.)
- Prepare 5- 500 ml bottles with 200 ml LB each.
- Autoclave to sterilize.
LB plates - Include 15 g agar with ingredients for 1 liter LB
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.
LBM (150 ml) - To a 150 ml bottle of LB add 150 ul 1M MgCl2 and mix well.
- Autoclave to sterilize (if ingredients used are not sterile)
LB top agar (400 ml)- 200 ml LB
- 200 ml 1.6% agar
- 400 ul 1M MgCl2
- Mix well and autoclave to sterilize (if ingredients used are not sterile)
LB+ Agar plates - Mix together the following pre-sterilized solutions:
- 200 ml 2X LB
- 200 ml 3% agar (melt agar in microwave first then add the other ingredients)
- 3 ml 40% glucose
- 0.4 ml 75 mM calcium chloride
- 0.4 ml 4 mM ferric chloride
- Pour as described for LB plates.
LB+top agar (1 liter)- Prepare as LB+ agar plates except use 250 ml 1.6% agar
- and 250 ml sterile deionized water
LMM medium (LB containing 1mM MgCl2 and 0.2% maltose) Use sterile ingredients:- 150 ml bottle of LB
- 1 ml 20% maltose
Large scale growth medium- Use sterile ingredients:
- 150 ml bottle of LB
- 150 ul of 1M MgCl2
- 300 ul of 1M CaCl2
- 375 ul of 40% glucose
1 M magnesium sulphate (1 liter)- Mix 246.5 g magnesium sulphate*7H2O with deionized water
- Adjust volume to 1 liter with deionized water. Autoclave to sterilize.
1 M MgCl2 (1liter) - Dissolve 203.31 g MgCl2*6 H2O in deionized water.
- Adjust the volume to 1 liter.
- Autoclave to sterilize.
Mussel glycogen (10 mg/ml stock) - Dissolve 10 mg of mussel glycogen (Sigma #61508 Type VII) in 1 ml of dH2O
- filter sterilize and store at 4 degrees C. Before use make a 1:10 dilution in sterile dH2O to give a 1 mg/ml dilution and store at 4deg.C.
3M potassium acetate - Dissolve 29.4 g of potassium acetate in 100 ml of dH2O
- autoclave and store at room temperature.
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
20% (w/v) PEG-8000 and 2.5M NaCl- 100 ml sterile 3M NaCl
- 25g PEG-8000
- Mix with heat if necessary.
- Adjust the volume to 125 ml.
Proteinase K (20 mg/ml stock) - Dissolve 20 mg of proteinase K in 1ml of sterile dH2O and store at -20deg.C.
- Before use make a 1:200 dilution in dH2O to give a 0.1 mg/ml dilution.
RNase stock solution - Prepare a 10 mg/ml solution in 10mM Tris-pH7.5 15 mM NaCl.
- Heat to 100deg.C for 15 minutes. Allow to cool slowly to room temperature.
- Add an equal volume of sterile 80% glycerol.
- Store at -20deg.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.
2.5X SDS-EDTA dye mix (10 ml) - Mix: 1.25 g Ficoll 400
- 2.5 ml 10% SDS
- 2.0 ml 0.5M EDTA
- 0.5 ml 1% bromophenol blue
- 0.5 ml 1% xylene cyanol FF
- Adjust volume to 10 ml with dH2O.
- Store at room temperature (SDS will precipitate at 4deg.C)
10 X SMO (1 liter)- Mix: 500 ml 1 M Tris H2O pH 8.0
- 58.4 g sodium chloride
- 20.0 g magnesium sulphate*7H2O
- Bring to 1 liter with deionized water.
- Autoclave to sterilize.
SM (1 liter) - Mix 100 ml sterile 10 X SMO with 900 ml sterile deionized water
SM+ (1 liter)- Mix 100 ml sterile 10 X SMO
- 5 ml 2% sterile gelatin and 900ml
- sterile deionized water
3M sodium acetate (100 ml)- Mix 40.8 g sodium acetate with 50 ml deionized water
- Bring volume to 100 ml with deionized water
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
X-Gal plates - Melt 200 ml 3% agar in a 500 ml size bottle
- Pour 200 ml 2 X LB into the 3% agar and mix well
- Add 0.8 ml dimethylformamide containing 16 mg X-Gal mix well
- Pour plates immediately; makes 12-16 plates
References:
Arber W. Enquist L. Hohn B. Murray N. and K. Murray. (1983). Experimental methods for use with lambda.In: Lambda II (ed. R. Hendrix J.Roberts F. Stahl and R. Weisberg) pp. 433-466. Cold Spring Harbor Laboratory Cold Spring Harbor New York.
CRI Laboratory Manual: RFLPs Project (1989).
Helms C. Graham M.Y. Dutchik J.E. and M.V. Olson. (1985). "A new method for purifying lambda DNA from phage lysates". DNA 4: 39-49.
Sambrook J. Fritsch E.F. and T. Maniatis.(1989) Molecular Cloning A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press.
