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Plasmid out of BL21 for mutagensis


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#1 TGS

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Posted 17 November 2010 - 12:56 AM

A short question which I seem to can't find an answer via google or manuals.
I have a plasmid I want to mutate. Right now I don't have the plasmid itself, but only a stock of the plasmid in BL21 cells. Now I'm wondering if the plasmids directly prepared from BL21 cells are good for site-directed mutagenesis or if I get a problem during the digest of the parent plasmid with DpnI?
I'm wondering since BL21 are dam+ but seem to have a function loss mutation of dcm. Do any of you had experiences with this? Is dam-methylation enough for a DpnI-digest?
A short answer would be nice, since then I could directly start the mutagenesis reaction or at least could directly re-transform in TOP10... and I wouldn't need to make a test digestion with DpnI to see if it works or not.

Thanks, Julian

#2 pDNA

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Posted 17 November 2010 - 09:00 AM

should be no problem in general since dam is responsible for the methylation of the A in the GATC-motif, therefore i expect no influence on DpnI-function.
The other thing i would worry about is the recA mutation in BL21(DE3) ...since the plasmids there tend to dimerize or even multimerize. I would just use that strain for expression and not for cloning or DNA isolation (it is endA as well ...therefore plasmid quality is affected as well).
If you wann do it quick and dirty ...do it the way you suggested it may work. I would prefere to do it the other way round using the original plasmid stock and transfer it in a suitable cloning host. But that is a matter of taste :)

Good luck!

Regards,
p

#3 TGS

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Posted 18 November 2010 - 08:25 AM

should be no problem in general since dam is responsible for the methylation of the A in the GATC-motif, therefore i expect no influence on DpnI-function.
The other thing i would worry about is the recA mutation in BL21(DE3) ...since the plasmids there tend to dimerize or even multimerize. I would just use that strain for expression and not for cloning or DNA isolation (it is endA as well ...therefore plasmid quality is affected as well).
If you wann do it quick and dirty ...do it the way you suggested it may work. I would prefere to do it the other way round using the original plasmid stock and transfer it in a suitable cloning host. But that is a matter of taste :)

Good luck!

Regards,
p


The amounts of plasmid extracted from and the quality of the plasmids from BL21 are fine according to my experiences. Once I had to prepare another plasmid and also only a BL21 Stock was available. Worked perfectly fine and the plasmid itself was also doing what it was supposed to do. But back then I just needed it for basic cloning stuff and not for mutagenesis, that is why I wasn't completely sure. Thanks for helping me out!

#4 TGS

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Posted 23 November 2010 - 06:39 AM

BTW I test-digested my plasmid and the digestion of the plasmids out of BL21 with DpnI works just fine.




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