Appendix I Commonly Used Recipes RECIPE LIST 1. Stock solutions for media preparation 2. VT and LB media and plates 3. TB plates for lambda phage 4. A plus B minimal medium for plates 5. M10 liquid minimal medium 6. Phage R17 and P1 plates 7. Phage P1 top agar 8. Phage T 4 plates 9. Phage Mu plates 10. Tetrazolium plates 11. EMB and EMBO plates 12. Xgal plates 13. Arabinose fucose minimal plates 14. Ribitol plates 15. Stab-agar for storing strains 16. Phage lambda suspension medium and storage buffer 17. TM medium for nitrosoguanidine mutagenesis 18. SOX TE buffer for DNA 19. 2X freezing medium for cells 20. 20X SSC buffer for DNA 21. Formamide 22. Carrier DNA 23. Distilled water 24. Chocolate chip cookies 1. Stock Solutions for Media Preparation Sugars are made at 20% (wtlvol). Amino acids are made at 1%. Vitamin B1! thiamine! is made at 1 mg/ml. MgS04! CaCI2! and MnCI2 are made at 1 M and FeCI3 at 0.01 M. 196 Commonly Used Recipes EDT A (ethylenediaminetetraacetic acid) must be neutral­ ized to a pH near 7 in order to be dissolved at concen­ trations up to 0.2 M. To neutralize 100 ml of 0.2 M, approximately 6.5 ml of 10 M NaOH or KOH will be required. It is useful to have both Na-EDTA and K-EDTA available. These solutions should all be sterilized by autoclaving. If they are in the convenient milk dilution bottles (Fisher 2-943-5), 10 min of autoclaving is sufficient. Generally, several ml of water evaporate during the autoclaving and this amount may be restored afterwards. Mark the meniscus with a magic marker before autoclaving and later add sterile water to restore the former level. 2. VT and LB Media and Plates These yeast extract-tryptone broth media are two of the most useful media for growing bacteria. Almost all E. coli mutants grow on them. These two commonly used media differ slightly in the amount of yeast extract and are probably interchangeable in all cases. Most E. coli strains will attain densities near 1 g/liter of medium when grown on YT. Bac­ terial yield is roughly proportional to the amount of yeast extract and tryptone added up to a level about 5 times YT. Usually between 20 and 30 ml are poured in 100 X 15 mm plastic petri plates. In humid weather these plates, like all others, require "curing" for a day or two until faint wrin­ kles appear on the surface. Absolutely smooth plates have a tendency to perspire when placed in the incubator. If, as is frequently the case, the plates must be used immediately, the curing may be hastened by leaving the lids off during the 20 min required for the agar to harden. Airborne contami­ nation is rarely a problem if subsequent incubations need not exceed about 3 days. To prepare a set of plates, each with different ingredients or different concentrations of an ingredient, it is usually eas­ iest to put the concentrated ingredient in the empty plate, pipette in 25 ml of the medium plus agar containing any other desired components, and mix with the end of the pipette. For 1 liter For 15 liters (1.33-strength) NaCi 5g 100 g Bactotryptone (Difco) 8 g for YT, 10 g 160 g for LB Yeast extract (Difco) 5g 100 g Distilled H20 1 liter 15 liters Dow Antifoam A 2 ml For plates, add 15 g agar (Difco) per liter. Recipe List 197 Plates should not be more than 2 days old for plating phage, and, if used for plating 4>80, the NaCI concentration should be lowered 10-fold. Instead of cluttering shelves with many varieties of top agar, we use TB top agar on YT plates. 3. IB Plates for Lambda Phage The glucose which is present in the yeast extract of YT or LB medium inhibits synthesis of the lambda phage receptor pro­ tein which is located on the surface of the cell. Thus, although YT plates are satisfactory for plating most mutants of lambda, some lambda mutants require the use of TB plates. Bactotryptone (Difco) 10 g NaCI 2.5 g MgS04 1 ml of 1 M Distilled H20 1 liter Agar (Difco) 11 g if poured in plates, 8 g if used for top agar Top agar is conveniently sterilized in milk dilution bottles and kept solid. It is remelted in a microwave oven and cooled to 48°C before use. 4. A Plus B Minimal Medium for Plates This is a basic minimal medium to which the desired carbon source, amino acids, vitamins, and antibiotics may be added. Flask A (1-liter flask) Flask B (2-liter flask) Na2HP04 7g Agar (Difco) 15 g KH 2P04 3g Distilled H20 500 ml NH4CI 1 g Na2S04 0.8 g Distilled H20 500 ml a. Autoclave. b. Mix flasks A and B. c. Add sugar to 2 g/liter (10 ml of 20%), 10 ml of vitamin B1 at 1 mg/ml, and desired amino acids at 20 JLg/ml. 5. M10 Liquid Minimal Medium This is a good liquid minimal medium. It differs from the clas­ sical M9 medium (Anderson, 1946) in the inclusion of MnH which is required by the enzyme, arabinose isomerase. 198 Commonly Used Recipes Na2HP04 7g KHl04 3g NH4C1 1 g NaCi 0.5 g FeCI 3 0.1 M, 0.3 ml (shake before using, as this reagent settles after autoclaving) 1 liter Autoclave, and when cool add the following sterile solutions: 1 ml of 1 M 1 drop of 1 M add to make 5 X 10-5 M Sa. 20 X Concentrated M 10 Na2HP04 140 g KH 2P04 60 g NH4C1 20 g NaCi 10 g Add 900 ml H20, mix, and filter through Millipore 0.45-~m filter (47 mm diameter). Then add the following: FeCI 3 0.6 ml of 0.1 M MnCl2 1 ml of 1 M CHCl 3 5 ml, for sterility of the stock After autoclaving a 20-fold diluted portion, add 1 drop of 1 M CaCI2 (optional) and 1 ml of 1 M MgS04 (not optional) per liter. 6. Phage R17 and Pl Plates Agar (Difco) 15 g NaCI 5g Bactotryptone (Difco) 8g Yeast extract (Difco) 1 g Distilled H20 1 liter Autoclave, and when cool add the following sterile solutions: CaCI 2 1 ml of 1 M Glucose 2.5 ml of 20% (wtlvol) 7. Phage Pl Top Agar Bactotryptone (Difco) 8 g NaCi 5 g Agar (Difco) 6.5 g Distilled H20 1 liter Just before use, add'CaCl2 to make 0.025 M. Recipe List 199 8. Phage T4 Plates Bottom Agar Top Agar Bactoagar (Difco) 12 g 6g Bactotryptone (Difco) 13g 10 g NaCi 8g 8g Na + -citrate 2g 2g Glucose 1.3 g 3g Distilled H2O 1 liter 1 liter 9. Phage Mu Plates Bactotryptone (Difco) 10 g Yeast extract (Difco) 5g NaCi 10 g Agar 10 g Distilled H2O 1 liter Make the plates 1 mM CaCI2 and 2.5 mM MgS04 by addition of sterile salts after autoclaving. For top agar, reduce the agar concentration by one-half. 10. Tetrazolium Plates These are the most convenient plates to use for the isolation of carbohydrate-negative strains. If the cells grow on the added sugar, then their colonies first are white, and over several days turn pink and eventually faint red. The cells that do not use the sugar produce colonies that begin as pink, turn bright red after about 18 h, and remain red. The color indications on these plates are stable upon prolonged stor­ age. Excessive crowding of the colonies inhibits the produc­ tion of the red dye by sugar nonutilizers. Such inhibition becomes a problem at densities of about 500-1000 colonies per plate. a. Combine 50 mg of 2,3,5-triphenyltetrazolium chloride (Sigma or Kodak) and 1 liter of distilled H20. b. Dissolve all tetrazolium. c. Add 25.5 g of Bacto antibiotic medium 2 (Difco). d. Autoclave and cool. e. Add 50 ml 20% sugar. 11. EMB and EMBO Plates On these plates carbohydrate non utilizers form pink colo­ nies and carbohydrate utilizers form colonies of a dark color. On these plates utilizers outgrow nonutilizers, thus allowing utilizers to be detected when 109 non utilizers and 100 utiliz­ ers are spread on the same plate. After 2 days the 100 uti­ lizer-positives will have grown through the confluent lawn of nonutilizer-negatives and can be purified. 200 Commonly Used Recipes EMBO plates are the same as EMB with the exception that they do not have any carbohydrates. Bactotryptone (Difco) 10 g Yeast extract (Difco) 1 g Agar 15 g K2HP04 2g Distilled H20 1 liter Autoclave, cool, and add 40 ml of 1% eosin yellow (Difco), 13 ml of 0.5% methylene blue (Difco), and 50 ml of 20% sugar. 12. Xgal (5-bromo-4-chloro-3-indolyl-P-D­ galactoside) Plates An insoluble blue dye is produced when Xgal is cleaved by ,B-galactosidase. This is a particularly sensitive indicator for low levels of the enzyme. Shortly before use, dissolve Xgal at 20 mg/ml in N,N­ dimethyl formamide. Add this to any desired plate medium just before pouring. 13. Arabinose Fucose Minimal Plates D-Fucose, an analog of L-arabinose, inhibits arabinose induc­ tion of the ara operon via araC protein. Mutants resistant to fucose are constitutive and can be selected on these plates. Autoclave 250 ml A plus B minimal medium, cool, and add the following: Vitamin B1, 2.5 ml of 1 mg/ml L-Arabinose, 1.25 ml of 20% (wtlvol) D-Fucose, 2.5 ml of 20% (wtlvol) 14.