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Loss of insert?


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#1 chen81ddtt

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Posted 28 October 2011 - 02:35 PM

Hi all

I have been experiencing some cloning problems, if someone could give some suggestions. I am trying to clone two PCR products individually (from different genes and both around 3kb) to a 7kb plasmid. I used SacI site for one, and XbaI for the other one. After restriction digest, CIP treatment of vector, and ligation, I got 2 to 3 on my vector only plates, and got 20 to 50 colonies on insert+vector plates. Things are going well at this step for both cloning.

When I mini prep them, and cut plasmids with SacI or XbaI, respectively. I can't get insert back, instead the plasmid got linerized. It seems like that plasmid doesn't contain insert, or one sites got destroyed during cloning?

I have used this plasmid to clone other big fragment at either SacI or XbaI sites, and all worked fine.

Does anyone has any idea why that is?

Thanks

CD

#2 pDNA

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Posted 28 October 2011 - 11:33 PM

SacI can be a tricky enzyme ...the most frequent problem encountered is salt inhibition ...so if your miniprep's contain residual salt than SacI will be inhibited.
For details see the [url="http://www.neb.com/nebecomm/products/faqproductR0156.asp#891"]NEB page[/url]!
Additionally, SacI cuts supercoiled DNA not that well so you need more enzyme ...see [url="http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_supercoiled_dna.asp"]here[/url]!

Is the plasmid size correct when it is linearized? (size=vector+insert?)

Overall i would go for a specific restriction enzyme that only cuts once in your insert ...and identify positiv clones that way!

Regards,
p




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