restriction enzymes primers - (Mar/10/2007 )
Hello,
I designed 5’ BamHI and 3’ XbaI primers, PCR of plasmid template, got the product (~1kb), cloned into pGEM-T Easy, cut with B/X, B and X as controls.
My X is not cutting, not even overnight digestion. I was told that maybe reverse primer did not work, but how came there was a product of right size then. Wouldn’t in that case Taq (used Taq) work of forward primer as far as it can and than after new annealing do the same. If there were fragment of various lengths I could get that but not ~1kb and vector only.
kajmak
XbaI has be inhibited by dam methylation. I am assuming this is the problem with your construct.
Methylation can explain why the vector is not cutting, but does not explain why the PCR product fails to cut (if it does not). There will be no methylation of the PCR product. Can you clarify if the cutting problem is with the vector or the PCR product?
I designed 5’ BamHI and 3’ XbaI primers, PCR of plasmid template, got the product (~1kb), cloned into pGEM-T Easy, cut with B/X, B and X as controls.
My X is not cutting, not even overnight digestion. I was told that maybe reverse primer did not work, but how came there was a product of right size then. Wouldn’t in that case Taq (used Taq) work of forward primer as far as it can and than after new annealing do the same. If there were fragment of various lengths I could get that but not ~1kb and vector only.
kajmak
Methylation can occur in the E.coli cells carrying your pGEM-T Easy plasmid, before you miniprep and digest. That's happened to me before. I had to design a new primer that didn't have the methylation site. What's the sequence of your reverse primer?
reverse primer is agatct and then gene, reverse complementary
pcr gene in bluescript, ligate into pGemt easy, transform e.coli, miniprep, digest, and then xba1 does not work, methylation in e.coli could be the answer but
because that did not work I cut gene out of pgem easy with ecor1, ligate into ecor1 digested pgem4z (which has xba1 site in mcs), transform e.coli (same strain), miniprep, cut with xba1 and it cuts, and the linearised plasmid is ~3.7 (pgem4z is 2.7)
why site in mcs wasn’t methylated?
the sequence beside xba1 determines if it will be methylated or not. so in MCS its made such that its will not be methylated. so xba1 should work cut rpoeprly in the MCS.