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plasmid, BAC, or cosmid? - (Sep/14/2009 )

Hi guys, i'm looking into cloning of a big chunk of genomic sequence of about 25kb to put into a vector and transfect it into some cell line. My question is will plasmid still work or do i need to shift to bac or cosmid? how big a sequence is considered too big for plasmid?


I don't really know much about BAC and cosmid and i can't seem to find out the info i need. So if anyone can also help direct me to some articles it will be really great.

Thanks in advance.

-krystle-

25 Kb is at the upper limit of normal plasmids. I would try a low copy number plasmid such as a pSC101 or p15 origin plasmid. Some of these contain a high copy origin intended to be removed as part of the cloning process. You might also consider the inducible copy plasmid pSCANS, which has the advantage that you can grow and propagate at low copy number and amplify at harvest with a high copy number to get more DNAk.

You could also consider a Bac or cosmid, but I think you'd find a low copy plasmid and a strain such as SURE or STBL2 will work, and be easier.

-phage434-

phage434 on Sep 15 2009, 07:58 PM said:

25 Kb is at the upper limit of normal plasmids. I would try a low copy number plasmid such as a pSC101 or p15 origin plasmid. Some of these contain a high copy origin intended to be removed as part of the cloning process. You might also consider the inducible copy plasmid pSCANS, which has the advantage that you can grow and propagate at low copy number and amplify at harvest with a high copy number to get more DNAk.

You could also consider a Bac or cosmid, but I think you'd find a low copy plasmid and a strain such as SURE or STBL2 will work, and be easier.



Hi, thanks for your reply. Is it possible for you to explain to me how does copy number of plasmid affect the size of insert i can put in? Also, is the cloning strategy for such large insert the same as typical cloning strategies? I frequently clone insert up to around 4kb.

-krystle-

I don't think it is well understood, but might be as simple as the amount of DNA in plasmid form vs. in chromosomal form. With a copy number of 400, there would be a lot of DNA from a 25Kb fragment.

-phage434-