what hybridization temp in pcr - (Jun/15/2006 )
i can't decide on something. i was earlier trying out this t/a cloning stuff, which was not successful, and so i decided to try directional cloning. since the previously used primers had given me correct sized dna (approx 950 bp), i ordered oligos that were really the same primers but with an xbaI site in the forward primer and a hindIII site in the reverse primer. both these sites naturally were introduced in the 5' end so as not to affect pcr priming. i intend to put the cdna in puc19.
the problem is with respect to their tm, which has gone quite high. the forward is 62.4 c (was 58.8c in the oligo without restriction site) while the reverse is 67.9 c (was 65.8c without restriction site), which is quite close to the extension temp of 72. i did a pcr today and maintained the original cycle which was melting at 94c, primer annealing at 52c, and extension at 72c. 30 cycles were given, with a 15 min final extension at 72c.
unfortunately, there has been no product. there is nothing wrong with the consumables since the control reaction (rna & primers provided by manufacturer) has the correct sized product. i feel the problem is the temperature. the rna i am using is the same that always yielded the cdna with the earlier primers.
incidentally, this was a one step rt-pcr, and initially the mix was maintained at 42c for 1 h to get the first strand.
so, given these primer tms, what cycle would you suggest.
thanks, and looking forward to your response.
Go up with the annealing temp. Try e.g. 60 or 65 °C. I have amplified things with a Tm of 76,5 °C. Worked. I think 52 °C can work, but is really probably too low.
If you already have a PCR product from your previous primers, you may be able to dilute that 1000x and use it as a template in your new reaction. I would not agonize over the Tm's. People worry about them far too much. If you continue to have problems, redesign your primers, rather than suffer trying to make these work.
thanks chalet and phage. i shall try out these.
by the way phage, these are primers that worked beautifully earlier, and are giving me the expected sized amplicon. all i did with this set was to add restriction sites, the remaining part is exactly the same.
so, if i redesign the primers, what can the BASIS of the redesign be?
Well, you can try to figure out what is wtong with a primer, and make it a research project in itself, or you can just design another one and get your job done. Personally, I give primers two tries: Once with an annealing temperature of 55C, and once with a gradient PCR covering 48-62. Usually things work the first time. Rarely, the second gradient gives conditions in which the primer works well. My next step is to redesign the primer and reorder it. I may think harder about things that can go wrong -- hairpins, primer-dimers, but in general changing the location of the primer solves the problem.
Primer3 can help, but usually I need primers in specific locations, and may change the length (extend on the 3' end). In your case, I would guess that introduction of the RE sites produced a hairpin structure in your primer, but I can't tell without a sequence.
hi chalet and phage
an update for info...
i had introduced xbaI and hindIII in the forward and reverse primers respectively, and as you know there was no product.
i checked the possibilities of hairpins etc, and found that while xbaI site in the forward primer was not so bad, the introduction of the hindIII site in the reverse primer had generated possibilities of almost 8 hairpins. at the time i had ordered these two primers, i had also ordered a reverse primer with an xbaI site, and this gave the theoretical possibility of only a couple of hairpins. anyway, when i tried with the two primers containing the xba site, i got the product. in effect, i did not have to "move" the position of the primer.
i still have to clone the pcr product, and so will post the result of that later.
also, i checked and found that introducing the hindIII site in the forward primer will not create problems (that i can foresee), hence i have ordered a forward primer with the hindIII site, so that i can regulate directionality.