Hello I am trying to amplify a stretch of Double stranded DNA (of 1.4 Kb) using A forward primer of 112 base pairs and reverse of 21 base pairs. The Tm value of the 112 basepairs long Forward is 91 deg. and that of 21 base pairs in 63 deg.
My question is which strategy would be better so that the 112 basepair long forward will get annealed properly. along with the 21 basepair long reverse.
and secondly i would like to know if I initially run the PCR at a temperature where only reverse will anneal, and later add the forward and increase the temperature nearly to 80 deg so that i get a full stretch.
The difference between forward and reveres primer should be within 3 degree to done PCR, but if exceed you go to touch down PCR, But I think the best choice involve redesign primer and if you obligate to this product size the better way using nested PCR
The only thing you can do here is run the PCR with the optimum annealing temperature for your reverse primer (Tm - 5 C) and hope that the forward primer won't bind nonspecifically. You may need to extend the annealing time as a primer this long may take longer to find its target.
112 bp is a truly egregious length for a primer. Why so long?
I think that you must re-disgn you forward primer. it's sooooo long with very high Tm. Also the difference betwen the two Tm is very large.
Dr. Andrew, I have tried performing the PCR at 55 Deg. but the strategy did not provide me any expected result.
Just a while ago before i am writing this, i have tried performing PCR intially for 10 cycles at 80 deg with PCR mix devoid of Reverse Primer, and later after 10 cycles, I have added the reverse primer and ran the PCR at 55 deg, I have got no result via this strategy. I have used it coz i felt that in intial cycle at 80 deg the long primer may bind and amplify for few strands and later after adding reverse primer (which ran for 30 cycles) the entire full stretch would be completed.
I have neither got non-specific nor any other bands, except very faint band near 100 bp.
Should i try doing a gradient from 55-75 deg with an interval temperature of 4 deg.? and see if its working.?
Dr. Basma
The 112 Base pair long is actually a two single stranded DNA's that were annealed and the double stranded DNA codes for a signal peptide and harboring restriction sites at both ends, I have ligated it to a gene (containing Restriction site at far end) and trying to perform the PCR with the strand (of 112bp) as forward primer and Gene specific reverse primer so as to amplify-elute the whole stretch so that i can clone it into a vector.. so as to express the signal peptide which would release the gene product into the extracellular matrix.
So intially i thought of giving a try to use the 112bp single stranded nucleotide as the primer, if it really doesnt work i will have to re design the forward.
In this case, I think you can ignore the signal sequence and calculate your Tm based on the part of the sequence that is homologous to the target.
However, I think in practice it would be easier to amplify the sequence with a normal-sized primer, possibly with a short tail to give you a convenient restriction site, and then ligate on your signal sequence; if necessary you could then reamplify using a forward primer homologous to the end of the signal sequence.
With an amplicon of 1400 bp you will of course need to extend the extension time to several minutes, but I'm sure you know this already. Also, it is a good idea to use special long PCR mastermixes as the standard mastermixes do not work well with very long amplicons.
Hi Goutham,
The extra long (>100 bases long) primers are usually for amplification of resistance cassette of marker for recombiniering proteins/genomes; or for tagging genes. These very long primers are always using only one tier of their 3`end during the first cycles, so the first 5 cycles are the most critical in your PCR.
I agree with Andrew, you should try as annealing temp the Tm-5ºC of your shorter primer (for your case it should be 58C). This rule works nice always.However, by my experience, I suggest to modify also the annealing time depending on the lenght of your expected PCR product size, let 10s per kb; but do not exceed the extension time.
hope these recomendations works for you,
Beatriz Sabater-Munoz
Dr. Andrew, did u mean to say that, I run PCR initially by adding Reverse primer alone at 55 deg and later add forward and run for few more cycles by increasing the extension time.?
I have used extension time inside the loop/cycle for one minute and final extension of 5 minutes till date.
Give me a day more, i shall try doing it at 55 Deg again as the last time i tried the template concentration was troubling me at few steps. This time i have confirmed it , so i shall re try doing at 55 Deg and i think extension with in the loop for 1 min and final extension for 5minutes will be fare enough for amplicon of 1.256 and 1.56 Kb.
No - there's no point in trying to run PCR with one primer. You don't get any amplification, just a linear increase in one of the strands. If this isn't obvious, it's time to pick up a textbook and revise PCR theory.
I don't think 1 min extension is anywhere near enough for an amplicon over 1000 bp. I would normally allow four or five minutes. Remember that the final extension is just to tidy up the ends of unfinished amplicons. This may seem a lot, but you have to allow for the problem of processivity - that is how many basepairs the enzyme can synthesise before it falls off. This will happen several times during synthesis of such a long amplicon.
Forget that. It's much more cost-effective to have the whole thing synthesized from scratch. Especially once you take into account indirect costs like your time, support staff time, facility... Just sit back for some days and let the synthesis company do the work for you. These days it's no more than a few 100 bucks for 1.4 kb.
I suggest to redesign your primers.The temperature is high and the base pair is also high.
Why do you need to use such primer? 112 base pairs is too much for a PCR!
Do I underestand right? IS Forward primer lenght 112 bP? How much is the PCR product size? 91 is almost denaturing temp. Shall you please check your F primer size and Ta?
I am sorry but this is not gonna work! You always get a high Tm when using such a log oligos. You must reduce the size of it. Why this length?
You have to recalculate the tm for longer primer according to base paired to your gene you can ignor the added bases
If you really need that length because you are constructing something, you could try to do this in two to three steps, e.g.:
1. Try the PCR with a primer that has only the 23 or so 3' basepairs of the 112 bp primer. Does it amplify? If not, check sequences, optimize etc.
2. Once that PCR works, try using a primer that has the 3' 50 bp of the 112 bp primer. Try the PCR both with the original template and the PCR product of step 1.
3. Once step 2 works, try amplifying the PCR product of step 2 with the original 112 bp primer. Alternatively, use a 60-bp primer that overlaps the primer from step 2 by 20 bp, and then another one that overlaps.
Of course this is a lengthy process - I assume that you have no other option and cannot afford commercial synthesis.
All the best
Gerald
Yes i am redesigning a smaller primer. Buy intially i wanted to try using a 112 basepair primer. But i have tried almost 5-6 strategies that were used in amplifying via long primers, neither of them worked ., may be coz of large Tm value of forward.
Thank you all for ur scientific suggestions
one thing you could try is doing the pcr with only one primer then using the products from that as template to run the pcr with the other primer.
Redesign your primer is a good idea. I also suggest more PCR cycles if you wanna amplify that big fragment.
Using such long primes for PCR is unusual. I've never heard about anything similar.
Long primer leads to very high Tm. So, at low Tm you'll have poor hybridization quality. At high temperatures (80-90) DNA polymerase will not work.
However, the Tm prediction algorithm usually not works for such long sequences. For example 95 deg. in PCR is enough to completely dissociate genomic DNA (more than 10 000 letters). Maybe 91 deg. is wrong estimation.
So, the best solution is to avoid such long primers.
If not, you'll need to experimentally check very broad hybridization temperature interval (from 60 to 85 deg). Maybe, without any results at all.
As far as I can understand, both ways lead to poor quality of PCR reaction.
By the way, is it absolutely nessessary to use such a long forward primer?
You could use a three primer aproach. One normal reverse primer of about 25 bp in lenght, second primer 25 bp specific to the target with 20 bp extra on the 5' part that functions as a flag that contains 26-45 bp of the 3' part of your long primer sequence, a third primer that contains the 26-45 bp flag together with the remaining 67 bp sequence of your long primer.
In this case the efficiency of your PCR is determined by primer 1 and 2.
The difference between forward and reveres primer should be within 3 degree to done PCR, but if exceed you go to touch down PCR, But I think the best choice involve redesign primer and if you obligate to this product size the better way using nested PCR
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