5 replies to this topic

[anonymous]

I have been trying to isolate PCR-able plasmids from yeast and so far have not been acceptable. I have tried using the CPG kit, the bio 101 kit, and the clontech kit, does anyone have any suggestions?

[anonymous]

maybe you can try to do PCR directly on the yeast, without plasmid extraction (see ref. Trends in Genetics 1990, Vol 6, p.236)

[anonymous]

If your plasmid is a shuttle vector, it can be transfered to E.coli, then you can extract the plasmid by general mini-prep. method.But if your plasmid is a low copy vector like cen-vector, you should cencentrate the totla DNA extract using ethanol precipitation before transformation to E.col.

[anonymous]

Sorry, in my first answer I did not answer your question! We always transform bacteria with plasmids obtained from yeast, and then do miniprep. For yeast plasmid extraction we use the following method:

Buffer A: 100 mM NaCl 10 mM Tris-HCl (pH 8) 1 mM EDTA 0.1 % SDS

1. Culture the plasmid-harboring cells overnight in 1 - 2 ml of medium. Cells with 1 - 4 OD600/ml are needed.2. Pulse 5-10 sec at 15'000 rpm (maximum speed) (in an Eppendorf tube).3. Throw away the supernatant.4. Resuspend in 200 µl of buffer A, keep cells on ice.5. Add glass beads just before the solution surface, mix with a vortex during 1 minute and sonicate.6. Add 200 µl of phenol.7. Vortex another minute.8. Centrifuge at 15'000 rpm for 1 minute.9. Throw away the phenol phase.10. Add 200 µl of phenol and reextract the same way.11. Take the water solution (about 200 µl) which contains the plasmids and treat with Glassmilk: add 600 µl of NaI solution and 5 µl Glassmilk suspension, put the tubes on the wheel for 5 min., then pellet the Glassmilk/DNA complex (pulse 5 sec), remove the supernatant and set aside, then wash the pellet 3 times with 300 µl New Wash, then pulse for 5 sec to remove the New Wash.12. Elute the DNA into water (20 µl) or TE buffer.

When not using the GeneClean-kit, another protocol can be applied:Steps 1 to 9 remain the same as above. Then:

10. Add 200 ml phenol/chloroform 1:1 and vortex.11. Centrifuge at 15'000 rpm for 1 minute.12. Throw away the phenol phase.13. Add others 200 µl of chloroform and reextract the same way.14. Take the water phase and adjust the volume to 400 ml with water.15. Add 40 ml 3M NaCl.16. Add ca. 1 ml of ethanol 100 %.17. Put the tube at -20 0C for about 10 min..18. Centrifuge at 4 0C for 10 min. at maximum speed.19. Throw away the supernatant.20. Wash the pellet once with ethanol 80 % and once with ethanol 100 %.21. Dry the pellet by putting the tube upside down on a Kleenex.22. Resuspend the pellet in 50 ml TE buffer.

Transform bacteria with 20 µl.

[anonymous]

You can use the EZNA Yeast Plasmid Extraction kit sold by Omega Bio-tek, Inc.It has worked well for me. You can go to their official website for protocol. I am sure this will help.

[anonymous]

You can use the EZNA Yeast Plasmid Extraction kit sold by Omega Bio-tek, Inc.It has worked well for me. You can go to their official website for protocol. I am sure this will help.