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PCR anomaly in a mid-range dilution of the template


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#1 gniknus

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Posted 15 April 2015 - 09:16 AM

Hi guys,

 

I'm trying to amplify cDNA using amplified cDNA as template (amplified using GeneRacer) and a number of gene-specific primer combinations. I've been having little luck in getting any products. Apart from PCR improvements such as Ta adjustments and PCR additives, I did a setup focusing on the amount of template, which produced some anomaly. The undiluted and the 100x diluted template both showed amplification in as shown by the presence of primer dimers and a tiny bit of the product. However, the 10x diluted template did not show any amplification, no primer dimers whatsoever, nada. Please look at the gel pic below.

 

A used undiluted template, B used 10x diluted template

150415.jpg  

 

and this is with 100x diluted template, primer dimers and some products showed up

150410.jpg

 

What might cause only the 10x diluted template to return no amplification? This is very puzzling for me..maybe you guys have some ideas? Thanks!



#2 phage434

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Posted 16 April 2015 - 04:39 PM

Pipetting errors. Have you tried simply reproducing this? Did you mix samples well?



#3 gniknus

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Posted 20 April 2015 - 02:29 AM

Yes, and this pattern was reproduced several times using fresh dilutions. Pipetting error might not be the case because the 10x and 100x dilution was made by means of dilution series.






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