PCR product dimer issue
Posted 13 April 2010 - 11:33 PM
Posted 14 April 2010 - 04:06 AM
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....
Posted 14 April 2010 - 03:58 PM
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.
Posted 14 April 2010 - 06:08 PM
Or maybe I shouldn't worry about all of that as it's product dimer....
How do you know that?
Posted 14 April 2010 - 11:21 PM
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
Posted 15 April 2010 - 08:46 AM
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.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 18 April 2010 - 03:39 PM
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.
Posted 19 April 2010 - 01:33 AM
Posted 19 April 2010 - 07:04 PM
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'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
Posted 03 May 2010 - 11:16 PM