As I wanted to amplify my GOI, I chose first 18 nucleotides including start codon in my forward primer. I add BAMHI recognition site at the beginning and 3 nucleotide extension for the efficient digestion. I did the same on the reverse primer. I chose 18 nucleotides including stop codon in my reverse primer. I add ECORI recognition site at the beginning and 3 nucleotide extension for the efficient digestion. However, the difference between Tm of primers are extremely high (20C) and GC content was %73 and %30 respectively. I tried gradient PCR for optimizing annealing temperature but could not obtain any band after agarose electrophoresis. I am using Q5 High Fidelity DNA polymerase and tried GC Enhancer solution in the kit but it did not worked. I could not change the majority of primers because I need the whole gene for efficient expression. What can I do for my experiment? How can I increase the efficiency of my primers?
You must use the annealing temperature calculated from only the speceific part of the goi to the primers. In the first few cycles you cannot use the BAMH1 and additional bases in yout Tm calculations so your primers are really only 18 bases long when calculating T annealing Also 18 bases is too short for primers. Polymerases make more errors at too low temperatures so the annealing temperature should be much closer to 72c so that the polymerase is always efficient and accurate so make your specific part of the oligo longer if possible
I had the same experience a few times, however, my target DNA was about 2 kb at that times, I had no experience amplify 2000 kb (2.000.000 bp?).
Anyway, what I have done was (1) simply check the direction of the primer (especially reverse primer) (2) check that the genome or template was there and not too much (I had experience fail to get band due to too much template), (3) make sure following the protocol of the master mix (I used primeSTARMAX).
good luck!
I think 2 kb was meant here.
When you are calculating the Tm, you should only do that for the nucleotides that are binding to your template, that is, the restriction enzyme cut sites or other additional nucleotides that are on the 5' overhang should not be considered.
From what are you trying to amplify the product? The template could be the reason why you are not getting a product, for example, if it is genomic DNA.
I would also try with a better matching another set of primers that gives a larger fragment and use the amplified fragment as template in another round of PCR with your current primers. Alternatively, you can try amplifying your construct in two fragments and combine them/clone by Gibson assembly. I love Q5 Polymerase and works mostly very robust for me but using another polymerase can also help.
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