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Old 05-16-2014, 07:00 AM   #1
Neiltje
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Location: Belgium

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Default primer dimer problem in PCR using Illumina protocol

Hi!
I'm using the two step PCR Amplicon approach to to some targeted sequencing.
My PCR with my primers without adaptor sequences works just fine. But when I add adaptor sequences according to the protocol :
Append to 5 end of forward PCR primer:
5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[locus specific sequence]
Append to 5 end of reverse PCR primers:
5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[locus specific sequence]

all of a sudden, I only have primer dimers in my PCR product.
Anyone ran into this problem? How did you solve it?
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Old 05-16-2014, 04:11 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

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This indicates that there is a primer heterodimer with strong bonds. First step would be to raise annealing temperature to favour your intended amplification while reducing unwanted primer-dimer interaction. If that is not helpful, next step would be to design new region specific primer(s) and analyse primer-primer interaction between your amplification primers (primers included with appended sequences) carefully to ensure that there is little interaction in your intended annealing temperature. You will need to take into account the salt concentration in your enzyme buffer as well.
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