3 replies to this topic

[Ameya P]

This multiplex PCR, which has been optimised and has been working fine, suddenly gave many unspecific bands for a sample. Thinking, that something must be wrong with the primers, I made a fresh batch of primers and repeated the PCR to get the same results. When the same primers, were used with the positive control, it worked well, so we know its not the primers.

I then, used the primers in individual reaction tubes instead of the multiplex. Although, the PCR worked well, and gave bands of the right size, a couple of primers gave larger unspecific products. The biggest amplicon size in this reaction is about 470 bp, while I got single bands about 600 bp and >1 kb too in a couple of tubes. The only explanation, I can think of is that the region where the primers bind is somehow duplicated elsewhere on the genome.

I would like to identify these PCR products. Could you suggest some methods to do that.
What I am thinking is gel purify these fragments and using the primers get them sequenced. Supposing they give identical sequence to the target region for most part of the electropherogram, how can I locate this region on the genome?

Thanks,
Ameya

[Ameya P]

Primer set Two gives more unspecific bands, but I have marked the ones I would like to find out more about.

Thanks for your help.

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[Adrian K]

Hi Ameya,

Just in case if you had yet to find out the problem...
sorry for not noticing this post earlier...

IMHO, it looks like your PCR machine temperature and gone off. I experienced this before for my multiplex PCR and after numerous troubleshooting only i realize the machine temperature was is not correct.

Also, maybe you had put in too much template...

All the best!

Rgs,
Adrian

[Ameya P]

Hey Adrian,

Thanks for your reply. I think temperature is not an issue cause other reactions seem to be working well. And its only this sample that gave these weird bands, while others have routinely given the usual bands.

Thats why I want to know, why this sample. Are there are repetitions of my primer sites else where on the genome? If yes, then how do I find them? I could probably use FISH and design a probe using these primers. But then, we do not have the equipment for that. Thats why, I posted it here, so that someone could  give me a PCR/ Sequencing/ gel based method....