Lambda Methods Media and Solutions

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.