| Yeast Media, Solutions and Stocks | 点击： 作者： 来源： 时间： 2007-04-11 本站论坛
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|  | | |
| L-tryptophan |
20 |
1 g |
2 |
| L-histidine-HCL |
20 |
1 g |
2 |
| L-arginine-HCL |
40 |
1 g |
4 |
| L-methionine |
20 |
1 g |
2 |
| L-tyrosine |
50 |
200 mg |
25 |
| L-leucine |
60 |
1 g |
6 |
| L-isoleucine |
60 |
1 g |
6 |
| L-lycine-HCL |
50 |
1 g |
5 |
| L-phenylalanine |
50 |
1 g* |
5 |
| L-aspartic |
100 |
1 mg |
10 |
| L-glutamic acid |
100 |
1 g* |
10 |
| L-valine |
150 |
3 g |
5 |
| L-threonine |
200 |
4 g* |
5 |
| L-serine |
400 |
8 g |
5 |
|
|
* Store at room temperature |
Solutions and stocks:
40 mM EDTA, 90 mM 2-Mercaptoethanol (2-ME)
| 40 ml |
0.5M EDTA, pH 8.0 |
| 3.8 ml |
2-Mercaptoethanol (12M stock) |
| 456.2 ml |
sterile ddH2O |
| 500 ml |
Store at room temperature. |
5X HPB (High Phosphate Buffer) (for 1 liter)
- 146.1 g NaCl
- 134.0 g Na2HPO4
- 9.3 g Na2EDTA
- pH to 7.0 with concentrated HCl
- Autoclave to sterilize
SCE (100 ml)
|
|
Final concentration |
| 2M sorbitol |
50 ml |
1.0 M |
| 1M sodium citrate |
10 ml |
0.1 M |
| 0.25M EDTA, pH7.0 |
24 ml |
60 mM |
| sterile ddH2O |
16 ml |
|
------ |
|
100 ml |
Filter sterilize and store at room temperature.
SCE/ DTT/ Lyticase (for 40 ml, prepare fresh each time):
- 40 ml SCE
- 300 祃 2 M DTT
- 5600 units Lyticase
SCE/2-ME/Lyticase (2 ml)
- 2 ml SCE
- 16 祃 2-ME
- 0.2 mg Lyticase
Mbo I Buffer
- 100 mM NaCl
- 10 mM Tris-HCl, pH 7.4
- 10 mM MgCl2
- 1 mM Dithiothreitol
- 100 礸/ml bovine serum albumin
Miniprep Lysis Buffer
|
|
Final concentration |
| 1M Tris-HCl, pH 7.4 |
5 ml |
50 mM |
| 0.5M EDTA, pH8.0 |
5 ml |
25 mM |
| 5M NaCl |
10 ml |
500 mM |
| 1M MgCl2 |
300 祃 |
3 mM |
| 12M 2-Mercaptoethanol |
25 祃 |
3 mM |
| Nonidet P-40 |
100 祃 |
0.1 % |
| 10% SDS |
10 ml |
1.0 % |
Large Scale Prep Lysis Buffer
| 0.5 M Tris-HCl, pH 9.0 |
2.5 ml 2 M Tris-HCl stock |
| 3% sarkosyl 3.3 ml |
10% sarkosyl stock |
| 0.2 M EDTA |
4 ml 0.5 M EDTA stock |
Adjust volume to 10 ml with sterile ddH2O.
1 mM Tris/1% Sarkosyl (for 1 liter)
- 1 ml 1M Tris-HCl, pH 7.5
- 100 ml 10% sarkosyl
- 899 ml dH2O
200 mM Tris/2X SSC (1liter)
- 200 ml 1M Tris-HCl, pH 7.5
- 100 ml 20 X SSC
- 700 ml dH20
Yeast prehyb/hyb solution (for 10 ml)
- 7 ml sterile dH2O
- 2 ml 5X HPB
- 1 ml 10% sarkosyl
Yeast Lysis Buffer
- 50 mM EDTA
- 1% sarkosyl
- 10 mM Tris-HCl, pH 8.0
- 1 mg/ml proteinase K
References:
Sherman, F., Fink, G. R., and J. B. Hicks. (1986). Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Brownstein, B. H., Silverman, G.A., Little, R.D., Burke, D. T., Korsmeyer, S. J., Schlessinger, D., and M. V. Olson, (1989) "Isolation of single-copy human genes from a library of
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