Donis-Keller Lab Manual, Department of Genetics in Washington University School of Medicine
http://hg.wustl.edu/hdk_lab_manual/plasmid/plsmid11.html
3% agar
Add 6 grams agar to 200 ml deionized water. Autoclave to sterilize.
1.6% agar
- 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.
(25 mg/ml)
Ampicillin Stock
- 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.
100mM ATP (200 ml) (200 ml)
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
- 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
- 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
- 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
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
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