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early ct value in ntc


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#1 harish

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Posted 25 May 2009 - 09:51 AM

i'm getting early ct value (around 20-23) for non template control (ntc) without any ct value for the other samples (including positive controls) tested. But the dissociation curve shows optimum tm value for the samples tested, though no amplification plot was observed for them. the tm value for ntc was 75 and for the known positive samples and test samples were 80 - 85, which i think are within the expected range. i'm using 5 prime realmastermix for my real time pcr. can anybody please point out what's wrong with my reaction...? if it was primer dimer, then the ct value should be obtained in all the samples tested, but in my case, only ntc is showing the ct value. the positive samples were confirmed in ordinary pcr. the annealing temperature of the primers was set at 53 degree celsius.

#2 naba

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Posted 28 May 2009 - 07:52 PM

Have u check ur water content? Is it the rnase free water or depc water?

#3 harish

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Posted 29 May 2009 - 12:59 AM

Have u check ur water content? Is it the rnase free water or depc water?


i'm using nuclease free water. i've taken all the possible precautions to avoid any foreign contamination....i've tried changing the mastermix solution...but the problem persists....the fluorescence (dR) never goes beyond 300-350...

Theoretically, sybr green should bind to any double stranded structures it encounter....in my reaction, the dissociation curve shows definite peaks of Tm in the expected range (80 - 84) , which indicates the product formation....still, ct value is not coming up...

i have no idea why this is happening....




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