insertion verification - (Jan/07/2009 )
Good morning!
I've a little problem..
I want to verify if my gene is inside the vector (vector + insert = about 6500 bp ). I'm quite sure that the gene is inside my vector but I've done a further verification.
I ve designed a Fw primer in the vector and the Rv one inside the gene..done the PCR..but I can't see the expected band.
Where the solution to my problem?
the question is: could depend on the length of the vector + insert, so secundary structures that don't permit to the primer to recognize the sequences??
Thank U.
Fralab
To clarify, are you doing PCR of your ligation, or of a colony you obtained following transformation? I would only do such a PCR on DNA following a transformation, or I'd do a RE digest of the DNA.
Regarding your questions about the PCR, are you seeing no bands, or a band of the wrong size, or a smear? You might have to play with the PCR conditions a bit
I'm not sure what you're asking in the last question. Secondary structures are a problem with ssDNA and RNA, but not dsDNA. Your vector and insert are both dsDNA, correct?
Regarding your questions about the PCR, are you seeing no bands, or a band of the wrong size, or a smear? You might have to play with the PCR conditions a bit
I'm not sure what you're asking in the last question. Secondary structures are a problem with ssDNA and RNA, but not dsDNA. Your vector and insert are both dsDNA, correct?
I'm doing PCR of a colony obtained following transformation and I cant see any bands. the plasmid with the insert are ds. My doubt regard to the length of the entire template. negative result could be explained with other answers?
Why don't you just do a RE digest to see if your insert is there? You say you are sure it is there already, a digest could confirm this.
As to why the PCR isn't working, it could be because of your PCR conditions. Have you used these primer pairs before in PCR? It can sometimes take a few tries before you get conditions that reliably work. Also, are trying to PCR just one colony? I always screen 8-10 following a ligation.
I'm not clear on what you mean you 'doubt the length of the entire template'. Surely this should be the length of your vector+insert. The size of the template is pretty much irrelevant in PCR. You can do PCR from whole genomic DNA, which is a hell of a lot bigger than one plasmid. Do you mean that you're not sure what size PCR product you should get? You can figure this one out if you know the plasmid map of your final ligated product.
As to why the PCR isn't working, it could be because of your PCR conditions. Have you used these primer pairs before in PCR? It can sometimes take a few tries before you get conditions that reliably work. Also, are trying to PCR just one colony? I always screen 8-10 following a ligation.
I'm not clear on what you mean you 'doubt the length of the entire template'. Surely this should be the length of your vector+insert. The size of the template is pretty much irrelevant in PCR. You can do PCR from whole genomic DNA, which is a hell of a lot bigger than one plasmid. Do you mean that you're not sure what size PCR product you should get? You can figure this one out if you know the plasmid map of your final ligated product.
what are you saying is correct but the plasmid plus insert is circular..so is possible that the vector with the insert could be pakaged in a manner that the primers couldn't recognize the sites..it is only a personal supposition, probalbyan un-correct supposition
Once you digest your vector, one way to pick the right band is to run on a gel, extract the band you need, ligate and then clone.
As to why the PCR isn't working, it could be because of your PCR conditions. Have you used these primer pairs before in PCR? It can sometimes take a few tries before you get conditions that reliably work. Also, are trying to PCR just one colony? I always screen 8-10 following a ligation.
I'm not clear on what you mean you 'doubt the length of the entire template'. Surely this should be the length of your vector+insert. The size of the template is pretty much irrelevant in PCR. You can do PCR from whole genomic DNA, which is a hell of a lot bigger than one plasmid. Do you mean that you're not sure what size PCR product you should get? You can figure this one out if you know the plasmid map of your final ligated product.
what are you saying is correct but the plasmid plus insert is circular..so is possible that the vector with the insert could be pakaged in a manner that the primers couldn't recognize the sites..it is only a personal supposition, probalbyan un-correct supposition
BTW, do you PCR your fragment of interest? In that case too, you can extract it from the gel, ligate and then transform
sometime the colony PCR didn't work. I always use both colony PCR and digestion, and if the colony PCR fails to show the bands, I extract plasmid and use it as the PCR template (it worked). I think in the condition of 94 - 95 degree as the initiation step of PCR, your vector will become relaxing, so it can't deter your PCR primers from doin' their job ^^, the points here are the proper annealing temperature elongation time.