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    • #7811
      Ammie
      Participant

      Hi! I know that you should use primers with about the same Tm, but theoretically is that really necessary? Can you not have two annealing temperatures, one for each primer and have the highest first?

      An example of what I mean. Primer F have a Tm of 79, and primer R of 65.

      And I use the following cycle:
      Denaturation 95 1 min
      Annealing 74 30 sek
      Annealing 60 30 sek
      Extension 72 1 min

      Is this not possible?

    • #73778
      ping
      Participant

      The difference is too big.If the annealing t is 74 and the Tm is 65 ,the primer won’t be that specific and may anneal not in the proper place.

    • #73779
      blcr11
      Participant

      You probably can do it that way, but you’re not gaining much by stopping to anneal at 74 C. The first primer should start to hybridize as soon as you get below its Tm and will keep on annealing as the temp goes lower. When you get below the Tm of the lower melting priimer, the annealation will go to completion. Just omit the 74 C anneal and you should be fine, I’d think.

    • #73782
      blcr11
      Participant

      Now that you’ve got both extremes for an answer–try protocols with and without the extra anneal and see if one is superior to the other. Unless you have real problems with non-specific hybridization–and especially if you take the trouble to anneal at both temps–I doubt the Tm diffs will give you that much trouble. It is true, though, that it is better to use Tm that are more balanced so you have better control over the annealling conditions. You may find you have to re-design the primers if there are problems with specificity. I don’t disagree with ping entirely. I just think think there’s a little more "wiggle-room" than ping does.

    • #73798
      mith
      Participant

      Setup a gradient and try for a temperature that allows you to have the best of both worlds. I’m working with primers designed for 63 degrees but I’m annealing them at 55 and they’re working quite nicely.

    • #73813
      LilKim
      Participant
      quote mith:

      Setup a gradient and try for a temperature that allows you to have the best of both worlds. I’m working with primers designed for 63 degrees but I’m annealing them at 55 and they’re working quite nicely.

      … there once was a time that I paid attention to Tm … but I do soo much PCR nowadays, that i’ve just used 55C routinely (for the past year)… and I haven’t had any problems…

      good luck!

    • #74172
      Ammie
      Participant

      Hi! This is what happened:

      I tried three different programs: The first one had two annealingsteps, the first one for the primer with the highest Tm and the second for the lower one. The second was without an annealingstep but with a longer extentiontime at 72 which also is the temperature in between the to primers Tm. The third one I did with an annealingtemp at 58, because I do almost all of my pcr at this temperature.

      The result: All of them worked very good. A large specific band and noting more!

      The template is plasmid-DNA, so it is much easier than "real" DNA.

    • #76726
      bluerose
      Participant

      Since this post was already started on the Tm, I’d like to ask a question about the Tm and annealing temperatures for my primers.

      I have 2 primers that I’m trying to hybridize to genomic DNA. So far I’ve had no luck in the PCR results. The Tm of my first primer is 61*C and the Tm of my 2nd primer is 58*C.

      From what has already been discussed here, is there a rule that you should use an annealing temperature 5 degrees below the Tm of the lowest primer?

      I have been testing my genomic DNA with one annealing temperature step and the temps have ranged from 58*C-61*C. I haven’t gotten good results yet and wondering if I should go lower in the annealing temperature step?

      Any suggestions??

    • #76728
      blcr11
      Participant

      Most definitely go lower than your lowest Tm. The idea is to go low enough to allow the primers of interest to anneal, but not so low as to allow non-specific hybridizations. In theory you can go to something like 1 degree lower, but if you try cutting it that close, I recommend you do a longish annealation time. Safer to go 3-5 degrees below your lowest-Tm primer. By doing the annealing at or above 58 C, I doubt your 58 C-Tm primer is hybridizing to any great extent.

    • #76731
      bluerose
      Participant

      so ranges of 53*C-55*C are good annealing temperature points?

      I am trying 54*C right now. It seems 55*C is a common PCR temperature. I may try that next if this run doesn’t show anything.

      I am using this pcr program, going through 30 cycles. I hold at 4*C at the end.

      94*C, 1 min
      94*C, 30 sec
      54*C, 1 min
      72*C, 1 min
      Go to step 2, Repeat 29
      72*C, 5 mins
      Hold 4*C

      Right now, I am testing various volumes of DNA template along with various annealing temperatures Do you think there is anything in my pcr program that I can modify to get better hybridization?

    • #76733
      blcr11
      Participant

      The program looks reasonable to me. It kind of depends on exactly what you’re doing and what enzyme you’re using whether the times make sense. As far as temperature is concerned, people use 55-65 C routinely–in some cases they will go even lower, but not typically. In your case, you should stick to temps less than 56 C, I’d guess. If your backgrounds are reasonable (and I’d guess they will be) then 54 or 55 C should be fine. If you still don’t get any product, try 50 C. But if 50 C doesn’t give product, then you may have a problem with either a poorly designed oligo, or a poorly prepared one, or maybe a problem with the enzyme or buffer condition (Mg vs Mn or no metals–that kind of thing can be a problem sometimes).

    • #76734
      bluerose
      Participant

      I am using Taq enzyme and Taq buffer with MgCl2 already in it from Invitrogen.

      I can try the other temps, but I’m hoping this 54*C works. I’ll let you know how it turns out. Thanks for the tips!

    • #76735
      blcr11
      Participant

      Taq should work if the desired product is short-ish. It isn’t the least error-prone enzyme around, nor is it the fastest. I prefer KOD Hot Start DNA polymerase from Novagen, but I think Invitrogen has similar high fidelity enzymes available.

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