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Yeast plasmid extraction - Molecular Biology - BioForum

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Yeast plasmid extraction


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5 replies to this topic

#1 anonymous

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Posted 30 July 2001 - 09:00 PM

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?

#2 anonymous

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Posted 01 August 2001 - 09:00 PM

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

#3 anonymous

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Posted 03 August 2001 - 09:00 PM

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.

#4 anonymous

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Posted 16 August 2001 - 09:00 PM

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 l 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 l of phenol.7. Vortex another minute.8. Centrifuge at 15'000 rpm for 1 minute.9. Throw away the phenol phase.10. Add 200 l of phenol and reextract the same way.11. Take the water solution (about 200 l) which contains the plasmids and treat with Glassmilk: add 600 l of NaI solution and 5 l 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 l New Wash, then pulse for 5 sec to remove the New Wash.12. Elute the DNA into water (20 l) 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 l 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 l.


#5 anonymous

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Posted 08 October 2001 - 09:00 PM

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.

#6 anonymous

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Posted 08 October 2001 - 09:00 PM

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.




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