Hi everyone,
I am having trouble in getting band with PCR. My forward primer is an oligonucleotide having 70 base pairs with Tm of 80.4 degree and the other one (Reverse primer) is of 38 base pairs with a Tm of 68.2 degree. The expected size of the fragment is 2920 bp. I have used the following condition but it did not work.
Prenature 94 degree for 2 min
Denature 98 degree for 10 sec
Annealing 60 degree for 30 sec
Extension 68 degree for 3 min
Final extension 68 degree for 6 min
(35 cycle)
Any suggestions about how to change the PCR setting in order to get the band?
Many thanks in advance.
You can do a gradient to check for the best annealing temperature,
and you can also change the MgCl concentration in your PCR.
the size is quite big that could also give problems in your PCR.
I have never tried gradient method. Can you provide me any link?
Its simple, you just try your sample on different annealing temperatures,
you're now at 60 so try 54,56, 58, 60, 62, 64
most PCR machines have a stepwise setup for gradient.
I would recommend a redesign of your primers. Two things jumped out to me.
1. Your primers are extremely long. Most guides recommend ~20 bp.
2. The annealing temperature of your forward primer is very high and too far from that of your reverse primer. Most guides recommend primers with melt temps around 60 C, with a difference of ~5 degrees between forward and reverse.
Here are links to a few helpful primer design guides:
https://www.idtdna.com/pages/decoded/decoded-articles/pipet-tips/decoded/2013/10/21/designing-pcr-primers-and-probes
http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
https://www.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html
your amplicone is more longer than 500 bp; I would recommend you different primers; you can make 5 different amplifications with overlapping amplicon.
Andrew is right. Your primers melting temperatures are too far apart and your primers are too long. I would advise designing new primers (http://simgene.com/Primer3) and making sure the melting temperatures are not so wide apart. Stick to
I would start by redesigning your primers, especially the forward primer, as suggested. I would agree with other comments and decrease your annealing temperature (optimize using a gradient) and perhaps increase your MgCl concentration to reduce the stringency. In addition, if you would prefer to amplify such a long amplicon in one reaction, I would try using a kit for long range PCR (ie Expand Long Template PCR System from Roche). Good luck!
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