PCR product dimer issue - (Apr/08/2010 )
As you can see from your own gel, reducing time did does "slightly" minor help.
Although I not sure about your total extension time per-cycle, I used to total eliminate the extension time by using only 2 step,
eg 95:5min, <95:1min, 55:1min>x30, 72:5min,4:store
i keep final extension longer for the "A" tailing for my TA cloning,if not I reduce to 1 minute or lesser...
I agree with Swanny, you can choose to cut off 5 cycles, or do a touchdown PCR.
If you want to really see what's happening, you could also do a gradient PCR, to nail when the secondary band starts to be an issue. I'm not at all convinced that simply playing about with the annealing time is an elegant or robust solution.
There must be something about the annealing conditions that causes this band to appear, so you need to consider the chemistry. This means binding kinetics, which is sequence-, temperature- and salt-dependent. Are you able to change the 3' end of the primer? what about the 5' end? You may then have to rework other primers for the sequencing reaction, but if it means you'll be able to confidently sequence any (or almost any) sample, it will be well worth the effort now.
I'm afraid that I can not change the M13 tag sequence which seems to cause the problem and I can not have other primers for sequencing than that M13
rudzielec on Apr 19 2010, 07:33 PM said:
In that case, work on the physical conditions of the reaction. Play around with the annealing temp. remember whatever it is that you have amplified, it doesn't amplify very efficiently, so you won't have to do too much to knock it out completely.
I've got the sequencing results but id doesn't look ok - apparently it's because signal is too strong....