LiOAc YEAST TRANSFORMATIONa
望天门山的古诗(e notes at end)
U STERILE TECHNIQUE throughout.
1. Inoculate a 10 ml overnight culture with a single yeast colony. If starting with a yeast strain with no plasmids, u YPD. If starting with a strain that already has one or more plasmids u liquid drop-out media lacking the appropriate nutrient.b Incubate at 30oC in roller drum at top speed overnight. b
2. Measure the OD陈柏宇600 of the overnight culture.c Make an appropriate dilution of the overnight culture into 50 ml of media in a sterile 250 ml flask so that the final OD600 is 0.2.
3. Incubate at 30oC, shaking at 200 rpm, until OD600 = 1.0
4. Transfer culture (using sterile technique!) to a sterile 50 ml polypropylene tube (Oakridge tube). Spin in clinical centrifuge at 3000 rpm, 2 min. This and all following manipulations are
done at room temperature.
5. Dump off media (quickly and carefully) and add 10 ml sterile water. Resuspend cells by swirling or gentle vortexing.
DNS服务器未响应是什么意思6. Pellet cells in clinical centrifuge at 3000 rpm, 2 min. Pour off water.
7. Add 5 ml of LiOAc/TE solution. Resuspend cells by swirling or gentle vortexing.
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8. Pellet cells in clinical centrifuge at 3000 rpm, 2 min. Pour off LiOAc/TE.
9. Add 0.25 ml of LiOAc/TE solution. Resuspend cells by swirling or gentle vortexing.
10. For each transformation aliquot 50 µl of cells to a sterile eppendorf tube. ALWAYS t up a control tube that will get no DNA.
11. Add plasmid DNAd. [For higher efficiency (e.g. for library transformations e) add DMSO to 10% (e.g. ~ 6 µl)]. Mix gently. Add 300 µl 40% PEG in LiOAc/TE. Mix by inverting two or three times.
12. Do not vortex. In the prence of PEG yeast cells are more fragile.
13. Incubate at 30oC for 30 min. Take this time to check the temperature of the 42oC, and warm and label plates. Remove cells to room temperature.
14. Incubate at 42oC for exactly 15 min. Remove to room temperature.
15. Plate 200-300 µl, including the bulk of the cells, onto appropriate plates. Incubate at 30oC with plates right side up. Turn plates over after 1 day.
NOTES
a This method is bad on the procedure of Geitz et al., 1992 Nucleic Acids Rearch 20:1425.
b When using YPD, 12 to 16 hours is sufficient for a 10 ml overnight culture. Growth in drop-out media may take much longer so the "overnight" culture may be started in the morning for the following day. Alternatively, a 50 ml overnight culture may be started - whi
ch may or may not need to be diluted the next day.
c Spectrophotometer measurements are only linear at OD600 between ~0.05 and 1.0. If the OD600 of the culture is over 1.0 it should be diluted 10-fold to take the measurement. Note: Ideally one should try transforming yeast at different OD600 from 0.7 to 1.5 to e which is best. Spects can differ, and this approach is easier than counting cells
d U no more than 1 µg plasmid DNA/50 µl of cells. For routine transformations, 5-10 µl of miniprep DNA or 0.1-0.5 µg maxiprep DNA is sufficient. For library transformations u 1 µg plasmid PLUS 30 µg carrier祝福的四字成语 DNA (such as Boehringer MB grade fish sperm DNA Cat# 1467140; e also Schiestl and Gietz (1989) Current Genetics 16:339-346).
e To perform 10 or more transfromations, scale up by using a 200 ml culture at step 2, and resuspend in 1 ml LiOAc/TE solution at step 9.
SOLUTIONS
得意造句子LiOAc/TE
For 50 ml, in a sterile 50 ml Falcon tube.
44.4 ml sterile water
5.0 ml sterile 1 M LiOAc, pH 7.0-7.4
0.5 ml sterile 1 M Tris HCl, pH 7.5
0.1 ml sterile 500 mM EDTA
40% PEG in LiOAc/TE
For 50 ml, in a sterile 50 ml Falcon tube.
40 ml sterile 50% PEG 4000
5.0 ml sterile 1 M LiOAc, pH 7.0-7.4
0.5 ml sterile 1 M Tris HCl, pH 7.5小宝宝生日祝福语
0.1 ml sterile 500 mM EDTA放松训练法
4.4 ml sterile water.
