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Problem with non-working primer

Technical question

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#1 Mohamed 1984

Mohamed 1984


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Posted 03 November 2014 - 03:54 AM

I am doing SYBR green based qPCR for HA gene of influenza. I have two questions:

1. I did a challenge experiment twice and in the first time, my primer (amplify 120 bp) worked nicely in different organs. When I repeated the experiment with the same virus dose and same conditions, I observed in a series of experiments that this primer did not give signals anymore even indifferent concentration (10, 7, 5, 2,5, 20, 30 pico mole) and in different annealing temp ( 55, 53)  while another primer (amplify 304 bp) gave nice ones. Could it be the case that in the second experiment there was less virus due to bad storage (for instance) and hence the low cDNA could be only detected with a primer (second) that amplify larger segment? What might be the other causes? 

2. Could I compare virus concentration in lung over 3 time points  using two different primer targeting different parts in the same gene ? i.e could I compare CT values from two different primers targeting different parts in the same gene. 



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