Removing vector restriction sites - and replacing with new ones (Jun/17/2006 )
i have a chimeric gene insert engineered by silent mutations with several useful restriction sites so i can exchange domains of the chimeric gene insert and test for effects. some of these restriction sites are at the multipe cloning site (mcs), i.e Bam HI and Xho I.
does anybody know of an easy method to remove these mcs restriction sites and incorporate a new one that is unique and not already engineered into the chimeric gene?
i dont want to have to use site mutant pcr and reampliy the whole plasmid again. or use Dnp I digests.
PCR your destination plasmid (1000x dilution as a template) with primers amplifying the plasmid backbone. Add or remove restriction sites to the 5' end of the primers as you wish (in your case remove everything, adding only BamHI and XhoI sites at the 5' end of the primers). Cut insert and PCR product with BamHI / XhoI, ligate, go. Bonus: little or no background.
i want to remove the Bam HI and Xho I sites from the vector but without pcr. i dont want to seuqnce the whole vector agin, right, cause it may have errors.
So what if it has errors. You care about the sequence of your insert, but not the vector backbone sequence. If it replicates and has the correct resistance, that is all you really care about. Your insert will be correct (or as correct as it would be otherwise) since it involves no PCR. I'll bet your existing vector has "errors" which you don't know (and don't care) about. Many of mine do, I know, having sequenced some of the them.
Cannot you just cut with 2 enzymes, one upstream BamHI and the other downstream XhoI? Then you could fill in (or check if the extremities are compatible) and ligate. But you would loose all the restriction sites between the two...
i've posted once a protocol that may hel you to do so.
idea : order two oligos. Once annealed they will form at 5' a part of the BamHI site and at 3' a part of the XbaI site. But after ligation of oligo and your vector, the sites will disappear.
in the exmple provided, the purpose was to change order of 2 sites, but mechanism is the same.
Same as fred suggested.
hey sage, no probs but my pi dosent want me to use pcr on the large vector. its an mfg retrovirus for use in clinical trials and must have no errors, for example in the LTR regions, which may affect expression of the transgene.
ok thanks fellas, ill look at this, cheers.