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Recommended competent cell for transformation of large plasmids?


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4 replies to this topic

#1 science noob

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Posted 19 March 2012 - 04:53 PM

Which competent cell type would you recommend for transformation of plasmid sized at 16 kb? My gene of interest is inserted into the pCrl empty vector.

From background reading, I have narrowed this down to JM109 and XL-10 Gold. Has anyone used either or both for large plasmids?

#2 leelee

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Posted 19 March 2012 - 06:19 PM

I've used neither- but we use either DH10B or DY380 (depending on application) for our BAC (which is about 230 kb).

#3 pDNA

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Posted 20 March 2012 - 10:25 AM

DH10B should be okay ...watch out for strains harboring deoR and hte mutations (GeneHogs, XL10-Blue, Stbl4, SURE). I would use electroporation ...this should give you better transformation efficiencies for larger plasmids!

Regards,
p

#4 Curtis

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Posted 20 March 2012 - 05:44 PM

This was my question too. I'm about to transform an 18 k plasmid, but my TOP10 can't take more than 10 K. I always thought DH10B is the source of TOP10. don't remember where I heard this....so according to you guys DH10B should be fine, and I will try electroporation.

#5 phage434

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Posted 21 March 2012 - 04:34 AM

The 10Kb is not an absolute limit. The efficiency of transformation for longer plasmids is low, but you should be able to get a transformant. There are strains designed for accepting longer fragments. I think Lucigen and Epicentre are two companies that come to mind.




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