I obtained very sharp bands in the region I am amplifying, but there are some faint bands below still (I guess primer dimers). I raised and lowered the temperature by one degree, for the +1degree nothing amplified whereas for the -1degree i saw more unspecific binding. What can I do?
I will use DMSO then. Don't you think that I will get unspecific binding with touch ? I use two temperatures higher and nothing amplified.
There can be many reasons for getting non-specific binding in PCR .So you can
Re-do the reaction with a negative control (no template). The non-specific bands could be from contamination of one of your stocks with foreign DNA (probably yours!). If this is a problem, use new stocks, always use autoclaved PCR vials
Redesign the primers and make the 3' longer. The extra bands may be from similar sequences to your target. Increasing the primer length will make them more specific for your target.
Increase annealing time if the non-specific products are shorter than your target. If they are longer than you target, reduce the annealing time.
Use less DNA template.
try these and you will get results
@Xiaojun Sun, @Anyebe
In my experience, using DMSO does help in PCR in certain cases But you might need to increase the amount of Taq slightly, as I found that DMSO seems to reduce the efficiency of Taq.
As for touchdown PCR, as I understand it you start off with higher annealing temperature to minimize non-specific annealing (at the cost of efficiencty), before lowering the temperature for efficient annealing and amplification. Perhaps that was a typo on your part, i.e. start with 60 for 10 cycles and then drop to 58.
A photograph would have helped.. if its faint, its trivial in case of conventional PCR. Its of no consequence if you are going to visualize only. But if sequencing, u can use gel purification. one thing you can do is reduce Mg2++ and then use two step PCR reaction, where you can run 10 cycles with annealing temperature at 5 degree Celsius higher than you Ta and then rest of the 20 cycles at original annealing temperature. This should solve.
Another thing, DMSO is very unpredictable, It reduces Polymerase activity. If it works at first go, its fine, otherwise its quite exasperating as you need to go on optimizing polymerase and Mg2++.
3 point, are u using conventional polymerase or hot start ??
As a general rule, as long as you are getting the band you want and the non-specific bands are not interfering, don't worry about them. You can spend months trying to optimalise them away. While you are doing this, the very products you are trying to avoid build up in the dust in your post-PCR lab and end up as potential contaminants - at which point no amount of optimalisation will get rid of them.
If you really need to get rid of the non-specific bands, I would suggest redesigning the primers. I try to make sure my primers end in a combination of A's and C's. Avoid G's and T's (particularly G's) as they are less specific due to formation of the non-canonical G:T basepair, which is almost as stable as A:T.
Of course, you should also check the primers for complementarity to each other and to themselves.
You may increase the annealing temperature to get rid of unspecific binding. Additionally, adding DMSO may also help.
But you should know first the reasons of unspecific product/multiple bands. your primers might not be specific enough or there is some other source of contamination while working. So be careful and always run a negative control. I hope this helps.
Regards,
Ansar
- Badges
- Science method