Bacterial Media Solutions and Stocks
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.
Alkaline Lysis Solution
| 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 |
Prepare fresh solution prior to use.
Ampicillin Stock (25 mg/ml)
Weigh out 250 mg ampicillin. Dissolve in 10 ml ddH2O.
Sterilize by filtering through a .22 micron filter and store at -20 degrees C.
When working with plasmids use 100 ug/ml in the LBM + Amp medium.
When workiing with cosmids use 200 ug/ml in the LBM + Amp medium.
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.
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
SOB + 20 mM Glucose
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.
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