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Poor efficiency standard curve

real time pcr pcr standard curve absolute quantification

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#1 detriar

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Posted 04 September 2013 - 12:13 AM

Hello all,

 

I have questions about standard curve for absolute quantification. I've already ran 2 times for this target gene, but I don't have any idea why the Ct between dilutions couldn't reach 3.3. From the picture I attached (please ignore Ct below 15), Ct between dilution almost reach 5 cycles! I almost sure it's not because of the pipetting (1:10 dilution, 2 ul template and 18 ul sterile ddH2O). Any chance primers caused it? Or any other aspects affecting the efficiency? I use 300 nM final conc. for each primers and 0.5 ng cDNA for 10 ul pcr final volume. I ran the housekeeping gene (the same method as this target gene), and the result is better (the slope -3.5). 

 

Any responds will be appreciated. Thank you :)

 

 

Cheers,

 

dee

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#2 Anpu

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Posted 06 September 2013 - 12:24 AM

Why don't you try different dilution? I was trying 1:10, 1:5 and 1:2 and the best was 1:5 (for me).

 

So maybe that could help.

 

Regards.



#3 detriar

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Posted 06 September 2013 - 02:33 AM

Dear Anpu,

thanks for the response! Yes I've tried 1:5 dilution and increasing each primer to 500 nM final concentration, and the result is better. Looks like I haven't optimized the reaction well before.

 

Thank you once again.

 

Cheers, 

dee







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