3 agar (200 ml) 1.6 agar (200 ml) Alkaline Lysis Solution Prepare fresh solution prior to use. Ampicillin Stock (25 mg/ml) 6.2M CsCl2 0.5 M EDTA pH 8.0 (2 liters) 40 glucose (1 liter) GTE solution 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: -- 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 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 -- 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 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. Add 6 grams agar to 200 ml deionized water. Autoclave to sterilize.
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
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.
10X CIP Buffer
Dissolve 42 grams of CsCl2 in 40 ml sterile 1X TE pH 8.0.
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 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
mix well and use sterile ingredients or filter sterilize stock solution volume 5 M KOAc 60 ml glacial acetic acid 11.5 ml ddH2O 28.5 ml 100 ml 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 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