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Strain requirements for routine M13 phagemid cloning?


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#1 dvliu

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Posted 29 March 2012 - 09:11 AM

I've been having trouble with what should be pretty straightforward digestion/ligation cloning on a phagemid vector for M13 phage display, specifically the pSEX81 plasmid. (http://www.progen.de...eets/PR3005.pdf). This plasmid has f1 and ColE ori's, and Amp resistance. The gene of interest is fused to the pIII phage coat protein. I'm making single clones, not a library.

I PCR'ed an scFv fragment with extension primers to provide two unique restriction sites (NotI/NcoI). The pSEX81 vector is cut with the same enzymes, gel purified (correct size insert is released), CIP'ed (antarctic), CIP deactivated, ligated (NEB T4 ligase), and transformed into One Shot Top10 chemically competent cells. I'll get 5-10 colonies on the vector+insert plate, and 0-1 colonies on the vector only, yet colony PCR comes out all negative, and sequencing gives me garbage (low sequence quality, mixed peaks). This vector is recently purchased, and sequencing of the original plasmid with the same primers gives great reads.

I have used the same general protocol with numerous other digestion/ligation cloning experiments with little to no problems. I have also cloned this same scFv fragment into other vectors without any problems (with different restriction sites) But for some reason this particular cloning experiment is giving me trouble.

In addition, this particular plasmid, original and unmodified, has been behaving strangely.
1) Colonies from an agar plate will sometimes not grow overnight at all, in either LB + Amp, or SOB + glucose + Amp. Sometimes it will grow fine in SOB + glucose, and little to no growth in LB. (16 hours at 37C). I've never had any problems with picking too small colonies or not enough of a colony with other plasmids/colonies
2) Occasionally, a miniprep will yield DNA detectable by absorbance but nothing will show up on the gel, even after loading what should be 2-3 ug. In these cases, 260:280 is a bit high, but not much, ~1.95.

****So here's my question: A methods paper describes the use of media with glucose when making libraries with this particular plasmid (http://www.ncbi.nlm....pubmed/21390868). As I mentioned, I'm only making single clones but even with glucose, I continue to have problems. However, this makes me think that perhaps there are other considerations for routine cloning with this plasmid. I've been using OneShot Top10 from invitrogen. I know when actually doing phage display there are requirements for what type of E. coli strain you use (eg. needs to have the F' episome). Are there any strain requirements to doing routine cloning with these types of phagemid vectors? Does anyone have experience doing routine cloning with phage display vectors, or even with this particular plasmid? Am I missing a key detail?

Thanks for any help you can provide.

#2 pDNA

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Posted 01 April 2012 - 12:04 AM

according to what i found on the internet ...the pSEX81 contains a lac wt Promotor derived from the pBluescript vector. Therefore, i would use glucose (1-2% in the growth medium) and a host strain that is lacIq (rather on the genome or on an f-plasmid) to prevent basal expression. Antibody fragments are bitchy ...and depending on what kind of fragment u are trying to express ...the cells will do everything to prevent expression ...and in combination with periplasmatic secretion (pelB leader) ...they situation gets sometimes worse.

Hope this helps!

Best regards,
p




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