C. Helms
10X CIP Buffer
- 10 mM ZnCl2
- 10 mM MgCl2
- 100 mM Tris pH 8.0
6.2M CsCl2
Dissolve 42 grams of CsCl2 in 40 ml sterile 1X TE pH 8.0.
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.
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.
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
- 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.
LBM (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. Note: LB medium is prepared without MgCl2.
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
- 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.
10X Ligation Buffer
For plasmid subcloning:
- 0.5 M Tris pH 7.4
- 0.1 M MgCl2
- 0.1 M DTT
- 10 mM ATP
- 10 mM Spermidine
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
- 3.0 ml 1 M Tris pH 8.0
- 9.0 ml sterile ddH2O
- 60 mg Lysozyme
- 12.0 ml - enough for 6 samples.
1 M MgCl2 (1Liter)
- Dissolve 203.31 g MgCl2*6 H2O in deionized water.
- Adjust the volume to 1 liter.
- Autoclave to sterilize.
85% 100 mM MgCl2 15% glycerol
- 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 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)
- Mix 98.15 g potassium acetate with 50 ml deionized water.
- Adjust volume to 200 ml with deionized water.
PP1
- 25% sucrose
- 50 mM Tris*HCL
- pH 8.0
- 1 mM EDTA pH 8.0
PP2
- 0.1% Triton X-100
- 50 mM Tris*HCL
- pH 8.0
- 60 mM EDTA
- pH 8.0
Shelf life of 3 months
PP3
- 30% polyethelyne glycol (PEG f.wt.8000)
- 1.5 M NaCl
RNAase (10 mg/ml)
- 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.
RNAase stock solution (5 mg/ml)
- 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.
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.
SOB Media
- 2% Bactotryptone
- 0.5% Yeast extract
- 10 mM NaCl
- 2.5 mM KCl
- 10 mM MgCl2
- 10 mM MgSO4
- 1.5% Agar (for plates)
SOC Media
3M sodium acetate (100 ml)
- Mix 40.8 g sodium acetate with 50 ml deionized water.
- Bring volume to 100 ml with deionized water.
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)
- 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.
Tetracycline Stock solution
- Prepare a 5 mg/ml solution in 100% EtOH.
- Store at -20 degrees C.
- Use at 10 ug/ml LB medium.
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.
