I run a touchdown PCR, and I found many bands in my no DNA template group and even other groups.In no DNA template group，there are 2 pairs of primers，and each primer is 20bp or 22bp. I run this again with new primers and sterilized objects, but I got the same result.Here is my result:
Dear Hua
If you have bands in your no template DNA group then this is either contamination of water buffer or primers and/or primer dimers. Try changing your batch of primers buffer and water then repeat
In addition look for primer (hetero) dimers with the following program
https://eu.idtdna.com/calc/analyzer
If you discover potential for primer dimer formation
Regarding non specific bands in addition consider the following
- non specific bands in my experience can sometimes be a problem in touch down PCR
- I would suggest sifting your touch down temp gradient 2C upwards in the first instance to see if eliminates the extra bands
- in general run your TD gradient from TM + 2 to Tm -3
- However Tm - 3 can sometimes not be stringent enough leading to extra bands
- I have had situations where I have followed the above rule but then adjusted my touch down gradient upwards: This has meant the difference between multiple non specific and usually a stronger specific band and just the stronger specific band respectively
- Is there any reason by the way you are using touch down; which is a good tool if the Tm of your forward and reverse primer are > 3C
- if < 3C I would try a temp gradient based on standard 3 step PCR in which you vary your annealing temp in 1C intervals from Tm-3 to Tm+2C; that is 6 x separate 3 step PCR reactions in duplicate plus water only control at Tm-2C
@Laurence Stuart Hall
Thanks for your reply. The size of the primer dimers is only about 50~100bp. In my no DNA template group (lane 8), however, most of the bands are >100bp. I wonder, could primer polymer be formed and amplified？Although I have run this again with new primers and sterilized objects and got the same result, this may be contamination of water buffer or primers. I will try again!
Thank you very much!
Dear Hua
- you can get oligoner structures but your most likely explanation is contamination and/or sub optimal PCR
- Thus change reagents; shift your touch down temp interval - which I assume is 5-10C - up by 2C and if primers are compatible perform separate standard 3 step PCR reactions varying the annealing temp in each reaction by 1C
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