PCR problem: long product (4 kb) from a cDNA library - (Feb/25/2006 )
hi, i hope someone can share some experience with this:
i'm trying to amplify a 4kb product from a mouse cDNA library. the nucleotide sequence from pubmed is longer than the ORF so i have my FW primer at the beginning of the ORF but my RV primers are about 100-200 bp behind the stop codon. i have one FW primer and 3 different RV primers just to make sure that i don't get a product because i've chosen a bad primer pair FW/RV (they all seem to be acceptable though), i use only one pair per PCR, of course.
then, tried already different annealing temperatures and starting with 52C and up to 60C (primers are 25-27bases long and have 7-9 mismatch nucleotides for introduction of a restriction site). TMs are all around 65C, no primer self pairs, no hairpins, only a bit of inter-primer pairing but within tolerated limits (around -5deltaG) - i've analysed all this with Lasergene.
tried it with two different polymerases: "herculase hot start" from stratagene and pfu from promega. at 52 and 55C i get some products around 1 and 1.5kb long. nothing comes above 3.5 which is my shortest product's size.
i do extension for 4.5 min at 72C. is this ok? do i need longer time for extension in the cycle? i make 30 cycles. i haven't played with Mg conc since i assumed so far it should be ok to use the 10x buff coming with the polymerases.
i suppose when the sequence behind the stop codon is listed in the pubmed it's not variable and my RV primers should be able to bind specifically there. could the cDNA be fragmented during the preparation and a whole-piece template is not available? - i tried two different mouse cDNAs - one from a cell culture mRNA and one from a mouse liver mRNA.
i tried amplifying also just a 0.5kb fragment of the same ORF and that worked fine. what could be the reason then?
do you guys have a suggestion
thanks in advance,
I tend to want to blame the cDNA library... how was your cDNA synthesized... from a poly T primer I guess? Was the 0.5 kb piece you amplified including the forward or reverse primer? I would guess it may be problems with synthesizing full length cDNAs?
Anyway I was involved with RACE cloning so that is why my tendancy is to blame premature stop... just something to consider...
one of the cDNA libraries was synthesized with a poly dT primer, the other one was made with a set of random hexanucleotides.
the 0.5kb fragment has its own two primers inside the ORF and i got this product out of the second library with the hexanucleotides.
so it might be an idea to combine my outside primers with the inside primers and first make to separate pieces which i'll have to put together later if it works...
oh, but with the oligo dT cDNA it shouldn't work i'm afraid.
anyway, 1min per kb is still ok with this size, right?