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help with qPCR

qpcr realtimepcr biology lab pcr

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#1 msc_student



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Posted 10 September 2014 - 07:02 AM

I need advice,
On my qPCRs my efficiency on my housekeeping gene is not good, but it is good on the target gene. These are both from the same standard mix pipetted twice. I asked some people in the lab and they said that maybe I need to use higher concentrations of the cDNA for the housekeeping primers to work, but I am running out of material so I found this forum and thought I would ask for advice before I continue. Could there be something wrong with my working solution of primers (the melting curve is fine)?

#2 Tabaluga


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Posted 10 September 2014 - 07:19 AM

The problem could be that the primers are not binding well to the (housekeeping gene) target sequence, so you could try and design yourself or look up in papers a different primer pair (primers are very cheap) before you waste your precious cDNA.

Alternatively you could try a different, unimportant cDNA at the same concentration and see if here again the housekeeping primers have a bad efficiency.

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Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.


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