When trying to amplify a 700bp gene by PCR, a got a band of 1000bp. My primers are about 60bp each. ???

Question

I should add that my gene is in a 3Kb plasmid. But here is the strange part. I extracted the gene by PCR before with shorter primers (20-25bp). I used both this PCR product and the plasmid containing my gene as templates in different reactions of course. Actually, I did 12 reactions, with a 10 degree temperature gradient (6 for each template). All of them produced the 1Kb band as the major product. This is what I really dont get. I wish it was non-specific binding, then I would know what to do.

Answer 1

It looks like you are getting non-specific products which means your reaction probably needs further optimization. Try one or some of the following:

- Decrease annealing time
- Increase annealing temperature
- Decrease extension time
- Decrease extension temperature to 62-68º C
- Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.
- Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
- Take less primer
- Take less DNA template
- Take less Taq polymerase

If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)

Answer 2

You're getting some non-specific binding. Try making the temperatures more rigorous and perhaps less cycles for the positive control and see if you get the right band. If not you may have to try a different set of primers. I always had to use two or three different primer combinations before I got it right. Sure looks nice the first time you get a nice clean PCR with a single band in the right spot!