PCR to produce 1.6kbp insert in fragments - (Oct/15/2007 )
I have been trying to amplify the ORF of my gene from its cDNA and I have been experiencing some difficulty. I now want to try and make it in 2 fragments by using different internal PCR primers and ligate them before cloning. I am just unsure how to go about it. What are the considerations that one must keep in mind while doing this type of PCR?
Thanks and any input is appreciated.
Do you really need to produce two fragments and then ligate? I think it would be easier to amplify the whole 1.6 kb ORF and then clone the entire fragment.
Hi SLAR,
I have previously posted in this forum about the problems I have been facing when I make the 1.6kBP insert and then clone it. I think I am missing a small part of the gene towards the 5 prime end, when I make the insert by PCR and clone it into the vector.I have confirmed this by expressing the clone and also by sequencing the clone and my PCR product. So one of the suggestions I recieved was that, try making it in two segments, ligate them and then use that for cloning.
I welcome any suggestion that you may have.
Thanks.
Hi priyas
Well if u want to amplify the complete Insert in two fragment,I suggest that you amplify it in such a way that the two fragment should have a overlapping sequence so that you digest it with a common restriction enzyme (this enzyme should have only one single site in complete seq) and then ligate them.This will ensure u dont miss out any region of ur seqeunce aswell the direction is maintained.I hope u understand what i meant to say.
Well if u want to amplify the complete Insert in two fragment,I suggest that you amplify it in such a way that the two fragment should have a overlapping sequence so that you digest it with a common restriction enzyme (this enzyme should have only one single site in complete seq) and then ligate them.This will ensure u dont miss out any region of ur seqeunce aswell the direction is maintained.I hope u understand what i meant to say.
Hi, I agree. The 2 sequences must overlap and have a restriction site in the overlap region.
Then is just a question of trying, I'm sure it'll work. Good luck priyas!
Hi cloned, SLAR, pernesblue and others who have posted their suggestions to help me figure out the issue.
Since the first 300 bp's of my gene are very rich in G's and C's and I tried PCR amplifying the entire open reading frame with and without 10% DMSO (5% betaine was suggested, but DMSO was just more accessible.). With the DMSO, I see a definite retardation of mobility on 1% agarose, indicating that the PCR product is longer than that obtained without DMSO. I have submitted the samples for sequencing and also trying to to clone it to see how that goes.
Thanks guys.