Donis-Keller Lab Manual, Department of Genetics in Washington University School of Medicine
http://hg.wustl.edu/hdk_lab_manual/yeast/yeast3.html
Purpose:
To rapidly isolate genomic DNA from yeast cells. Up to 24 YAC clone samples can be processed in one day as opposed to sucrose gradient preps which require 3 days. This method is designed for < 1 g wet weight of cells. Because care is not taken to prevent shearing of the DNA, the average size of the DNA fragments is probably less than 100 kb.
Time required:
Special Reagents:
- Lyticase (Sigma #L 8137)
- Nonidet P-40(NP-40) nonionic detergent (Sigma, Cat.#N-3516)
Procedure:
Day 1
- Inoculate 5 ml AHC or YPD with yeast colony of interest, then incubate at 30 degrees C for 1day if grown in YPD or for 2 days if grown in AHC medium.
Day 2
- Pellet cells by centrifuging at 2000 rpm for 10 minutes in J-6 centrifuge. Wash pellets twice in 10 ml of 40 mM EDTA/90 mM 2-Mercaptoethanol (2-ME), discarding the supernatant.
- Add 2 ml of SCE/2-ME/Lyticase mixture (see recipe below), and incubate at 37欳 for 2 hours to produce spheroplasts.
- Centrifuge spheroplasts at 2000 rpm for 10 minutes in J-6 centrifuge, then decant the supernatant.
- Add 700 祃 of lysis buffer (see recipe) and incubate at 68欳 for 15 minutes; vortex the sample intermittently during this time.
- Vortex to resuspend spheroplasts, then split this volume (approx. 1.2 ml) into 2 microcentrifuge tubes. Extract twice using 600 祃 of Phenol:Chloroform:Isoamyl Alcohol (1:1:0.02) by centrifuging at 12000 rpm for 10 minutes after addition, and recovering the upper, aqueous phase for the next round.
- Add 700 祃 of isopropanol to second aqueous phase; incubate at room temperature for 15 minutes, then centrifuge at 12000 rpm for 15 minutes. Discard the supernatent, then rinse the pellet with 70% ethanol. Air dry pellet or dry briefly in the vacuum concentrator.
- Suspend the DNA in 300 祃 of TE. Add 30 礸 RNase (0.05 units/mg). Incubate at 37?for >2 hours, with intermittent vortexing to dissolve the pellet.
- Pool the two microfuge tubes for each clone together, then add 600 祃 warm isopropanol. Incubate at room temperature for 15 minutes, then centrifuge at 12000 rpm for 15 minutes. Wash pellet in 70% ethanol, then air or vacuum dry.
- Resuspend in 300 祃 of TE, then add 750 祃 ethanol. Chill in dry ice bath for 10 minutes, then centrifuge at 12000 rpm for 15 minutes. Discard supernatent, wash in 70% ethanol, then air or vacuum dry.
- Resuspend in 200 祃 TE, then measure the DNA concentration on a spectrophotometer or by running out on a gel with standards. Expect approximately 20 礸 of DNA per 0.1g wet weight of yeast cells.
Solutions:
- 40 mM EDTA, 90 mM 2-Mercaptoethanol (2-ME)
| 40.0 ml |
0.5M EDTA, pH8.0 |
| 3.8 ml |
2-Mercaptoethanol (12M stock) |
| 456.2 ml |
sterile ddH2O |
| -------- |
|
| 500ml |
Store at room temperature. |
- SCE
| |
|
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/2-ME/Lyticase (2 ml)
2 ml SCE
16 祃 2-ME
0.2 mg Lyticase
- Miniprep Lysis Buffer
| 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 % |
| |
------ |
|
| bring volume to |
100 ml with sterile ddH2O. |
|
Filter sterilize and store at room temperature.
References:
Eric Green, pers. comm., from his protocol "Purification of genomic DNA from filters of yeast clones", 1989 (M V Olson lab).
上一篇：High Molecular Weight Yeast Liquid DNA Preparation 下一篇：Yeast Media, Solutions and Stocks
