24 replies to this topic

[rudzielec]

I can't reduce Mg concentration because it's already in a master mix..... I've tried lots of enzymes and that one which I use now is easy and always work for for those genes which I need to amplify now. Hmmm it used to work giving nice single band when I was using untagged primers. I'll give a try today Adrian, and I hope it'll work as I'm really fed up with that M13 tag :/

[rudzielec]

I've tried to reduce annealing and extension time. I tried to amplify 2 exons:
A - annealing as normal, extension reduced by 15s
B - annealing as normal, extension reduced by 30s
C - annealing reduced to 15s, extension as normal
D - annealing reduced to 15sl, extension reduced by 30s

Should I keep annealing time reduced to 15s and keep reducing extension time?? But the correct band is getting weaker... Or maybe I shouldn't worry about all of that as it's product dimer so the sequence will be the same but let say longer....

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[swanny]

How many cycles of amplification are you doing? Given the strength of the main product you could cut 5 cycles off.

Have you tried touchdown PCR? It might help you swamp out the secondary reaction.

I'd still take a few samples with varying amounts of the larger band and see what the sequencer makes of them.

[HomeBrew]

rudzielec, on Apr 14 2010, 08:06 AM, said:

Or maybe I shouldn't worry about all of that as it's product dimer....

How do you know that?

[rudzielec]

What else could be there if it's always double size of correct PCR product. I've tried to amplify my stuff with normal primers and then I used as a template product of first PCR and amplify that with M13 tagged primers and I still got that....
If it'd be sth like unspecific binding to the template (gDNA) it'd be always the same size doesn't matter which exon is amplifying..I think...
I'll probably give some samples to be sequenced and we'll see if that extra band makes actually any difference. So fingers crossed

[Adrian K]

Dear rudzielec,
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.

Good luck.
Adrian

[swanny]

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.

[rudzielec]

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

[swanny]

rudzielec, on Apr 19 2010, 07:33 PM, said:

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

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.

[rudzielec]

I've got the sequencing results but id doesn't look ok - apparently it's because signal is too strong....