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Reproducible Non-Specific PCR Product

PCR Sequencing Cloning

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#1 Epigeneticist

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Posted 20 May 2013 - 04:56 PM

I am genotyping mouse cells after cre-mediated knockout. The gene has LoxP sites with intron 1 and intron 10, so a large portion of the gene is deleted. To detect deletion by PCR, forward primer anneals within intron 1 and reverse intron 10, which gives a 600bp product. However, I also get a non-specific product. Genomic DNA from control (floxed sample, no knockout) and gene knockout samples both produce a product that is around 660bp. I sequenced the PCR product and the results are confusing (gel purified band, TOPO TA cloned and sequenced).

Clone 1: sequence at 5' end was the reverse complement of the 3' end - i.e. my reverse primer

Clone 2: sequence at 5' end was forward primer and 3' end was reverse primer. Seems correct, but blast results show sequence is similar to some mouse cloned product.

Clone 3: sequence at 5' end was the reverse complement of the 3' end - i.e. my reverse primer


The sequences matches the primers are 100% correct but not sure what the sequence is between the primers. The sequencing peaks look clean, sharp and good height. All sequence are the same size. Clone 1 is about 98% similar to clone 3 but both have no similarity to Clone 2. My non-template control does not produce anything.

No idea where to start, except to start over?

Sample problem - mRNA contamination?

Primer issues? Primers are reported in the literature.

Cloning or sequencing issue?

Thanks!

#2 pcrman

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Posted 21 May 2013 - 11:10 PM

How long are the sequence between the primers when you sequenced them? For any sequence you got from sequencing, can you try to blast them against other species such as human to see if they are contamination? I guess you either have contamination of your PCR system (in this case, you should discard everything and start over), or have non-specific binding to other location(s). Try do a in silicon PCR using your primer sequences to find out. For this, you can lower the criterion for Min Perfect Match.

#3 Epigeneticist

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Posted 22 May 2013 - 06:36 PM

I believe it is non-specific annealing. The primers and cells I am using are from the same paper, and I got the cells from the lab that published this paper. I cross referenced the primers in this paper and it appears the the forward primer that I am referring to is missing a cytosine at the 3' end. I am not sure if this was a typo or not, but it is one of many errors I have come across in this paper. I ordered the correct forward primer so hopefully it works better.





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