I'm doing PCR with primer tagging with attB1 and attB2 sequences for gateway cloning system. My target gene was ligated to pRS416 plasmid, 2 kb and 1 kb in sized. I'm using KOD FxNeo PCR kit and Tm for the primers are 70C. I have tried several Ta, ranged from 62 - 68 C with annealing time = 2.30 min but no PCR was observed. Then, I have tried touch down-PCR with Ta ranged from 55 - 65 c but again, no PCR product. What should I do to overcome this problem. Thank you.
The Tm of 70 is unusually high. Is this the Tm of the sequences of your primers that hybridize with your gene? Annealing time also looks very unusual, 2.3 min. Are your primers unusually GC rich?
My primers contain attB1/2 sequences for GATEWAY cloning, thus, their Tm are quite high.
But then you can't use that Tm to setup your PCR. Please calculate your Tm based on the nucleotide sequence that actually hybridizes with your target DNA.
But I have tried Ta = 55 c in Touchdown-PCR before, no PCR product was observed.
Well, maybe you have to try even lower Ta. Would you please re-calculate the Tms of the forward and reverse primers (minus flanking sequences) and let us know. How many nucleotides hybridize to your gene of interest?
Is 70°C the predicted Tm of your primers?
You should calculate it by taking into account the PCR conditions of your system, including Na and Mg concentration as well as DNA and primer concentration
the oligo analysing tool at Integrated DNA Technologies website might help
What is the length of your primer that anneals the cDNA sequence? It is very strange that you don't have any product, not even smearing.
You can calculate the Tm by a formula:
Tm=61.25+[0.41*(% GC/Totale)]-(500/Totale)
and you will try a PCR with a range of 8 temperature of annealing and through an agarose gel you check the PCR product.
For me 2.3 min for the Tm is so much....In my PCR using min 30-45 sec for the annealing time (repeat 30-35 cycles).
Are you doing 2-step or 3-step cycle PCR ?
For this kind of PCR the primer should be with a high Tm.
After a predenaturation at 95 C - 3 to 5 min. Try to use the 2-Step cycle : Denaturation at 95 C for 20 sec ; Annealing / extension at 68 - 70 C for 30 sec/1 kb).
Avoid to use dNTP from other kits, avoid primer dimer formation and be sure that there is no RNA in your sample, so use RNase in your DNA preparation.
It should work since you have a small fragments (1 and 2 kb), We have amplified a 23 kb fragment using this kit. and using a 2 step cycling with a tm equal to 70C.
Primers with high Tm values are usually very efficient. Do you have the correct primer sequence?
Annealing temp should be around 68-70ºC as Hafid Soualhine mentioned. Check your primers for hairpins and heterodimer formation using oligo dt analyzer website, https://www.idtdna.com/calc/analyzer.
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