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.
(1Liter)
1 M MgCl2
- 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.
(200 ml)
5 M potassium acetate
- 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.
(1 liter)
TE
- 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.
(1 liter) (1 liter) (2 liters)
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