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standard curve/ PCR efficiency problems...


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#1 fibonacci

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Posted 02 February 2010 - 07:55 PM

hi...

i face some problems with my experiments...
i have designed several set of primers for my target gene...
at first, i got multiple bands for my pcr product (using normal RT-PCR)..
then, i tried to optimize the annealing temperature & primer concentration but i still got the same result..
for your info, i'm purified my RNA and check it with Bioanalyzer chip...there is no genomic contamination and the RIN no is 9.3..
so, i think that the RNA quality is good...

then, i'm designing new set primers (1 of it lay at the exon-intron region)....
then, i do the RT-PCR...luckily, i manage to get specific band for the exon-intron primer but for the other set of primer, i still got the multiple bands...

so, then i proceed with qPCR experiment using the exon-intron primer...
at first, i run the standard curve to test the PCR efficiency for that primer (exon-intron).
i'm not running it in duplicate or triplicate..just 1 reaction using 10-fold dilution (100ng decreases to 0.001ng)..
unfortunately, i didn't get the efficiency between 0.9-1.1...for your info, i got the PCR efficiency = 1.75...
then, i repeated it using the new 10-fold dilution..but, i still got the PCR efficiency - 1.62...
then, i repeated the experiment but now i'm using electronic pipet from the dilutions steps until doing the master mix for qPCR experiment..

but i still got the same results...pcr efficiency > 0.9-1.1....

so, can anyone help me??
what's wrong in my experiment???
do i need to design new primer because it is hard to get the specific pcr product for this gene..

thanks..

#2 chrisbelle

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Posted 02 February 2010 - 10:17 PM

so, then i proceed with qPCR experiment using the exon-intron primer...
at first, i run the standard curve to test the PCR efficiency for that primer (exon-intron).
i'm not running it in duplicate or triplicate..just 1 reaction using 10-fold dilution (100ng decreases to 0.001ng)..
unfortunately, i didn't get the efficiency between 0.9-1.1...for your info, i got the PCR efficiency = 1.75...
then, i repeated it using the new 10-fold dilution..but, i still got the PCR efficiency - 1.62...
then, i repeated the experiment but now i'm using electronic pipet from the dilutions steps until doing the master mix for qPCR experiment..

but i still got the same results...pcr efficiency > 0.9-1.1....


Firstly, what is the machine and software that you are using for your real-time PCR?
I suggest you read the manual carefully. Does the machine assume "1" or "2" as 100% efficiency? If your machine assumes 2 as perfect efficiency then you're quite alright.

Multiple bandings indicate unspecific binding. You have to redesign primers with a more stringent Tm, and higher Ta.

Chris
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