Greetings
I am doing Aflp, for my pre selective amplification step i am using two primers Mse1+1 base and Ecor1+1 base. But both the priners have different annealing temp 52 and 55.
I tried gradient pcr but the results in selective pcr varies, i got different bands at different annealing temps.
Can anyone help/ suggests me the best anneling temp.
Hello,
you are absolutely right that the Tm for primers depends on their sequence and since you use two different primers they will be characterized by two different Tm. However, in AFLP procedure you are forced to use different combinations of primer so you need to standardise the method.
In my lab we followed the original procedure by Vos et al. 1995. According to that procedure the preamplification for all kind of primers (all combinations) having none or one selective nucleotide was performed according to following conditions:
Initial denaturation in 95°C for 1 min
and 30 cycles of
denaturation in 94°C for 1 min
annealing in 56°C for 1 min
elongation in 72 °C for 1 min
and final elongation in 72°C for 5 min.
In selective amplification we also followed the Vos et al. 1995 and used decreasing temperature of annealing (-0.7°C per cycle) in first 12 cycles starting from 65°C and finishing in 56°C for 30s, followed with 28 cycles in which annealing temp was 56°C for 30s.
Below I am sending you the link to the original paper by Vos et al. 1995 just in case you do not have it. Just have look at the page 4408, the paragraph entitled AFLP reactions.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC307397/
I hope you will be satisfied with your results.
Good luck
Piotr
Dear @piotr Androsuik, thank you for your suggestion, i am. Exactly doing the same but i am worried about the pre selective pcr which annealing temp to follow,bcz we have to chose only one best anneling temp. We can't use different annealing temp fir different combinations.
I also did pre selective with 56 anneling temp but my band in selective are different when compared with 50c.
Hi,
I am aware of the fact that applying of different annealing temeperatures may alter the obtained band profile. But here you need to choose one, standardised methodology for whole experiment, which you will apply for all PCRs.
Therefore we decided to follow strictly the method by Vos et al. 1995 and applied the same annealing temperatures for all primer combinations: 56°C in preamplification and in case of selective amplification decreasing temperature of annealing (-0.7°C per cycle) in first 12 cycles starting from 65°C and finishing in 56°C for 30s, followed with 28 cycles in which annealing temp was 56°C for 30s.
If you still have doubts have a look on other papers using AFLP method to check for the authors approach for that issue.
Best wishes
Piotr
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