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High Ct value - what to do next? - (Apr/22/2010 )

Hi,

I need to validate my primer set so recently i constructed a standard curve (1:5 dilution) from 40ng to 0.064ng/ul cDNA template, and the primer efficiency is about 109%. Problem is i have high Ct value, even from 40ng/ul the Ct is about 30, which is very high. The literature suggested to use higher template amount but i also read that the the amount of template used in qpcr should not exceed 50ng/ul. What could be the problem? Should i design new primer or should i try smaller dilution?

Thanks.

-wntiong-

wntiong on Apr 23 2010, 11:26 AM said:

Hi,

I need to validate my primer set so recently i constructed a standard curve (1:5 dilution) from 40ng to 0.064ng/ul cDNA template, and the primer efficiency is about 109%. Problem is i have high Ct value, even from 40ng/ul the Ct is about 30, which is very high. The literature suggested to use higher template amount but i also read that the the amount of template used in qpcr should not exceed 50ng/ul. What could be the problem? Should i design new primer or should i try smaller dilution?

Thanks.


What exactly do you mean by a high Ct value? i mean the number is??!!! and 40 ng is more than enough to se if the primers are fine.. i think u will have to take a peek at ur primers but before that into your amplification conditions and buffer primer mg+ dntp concentration etc!!
Best luck!!

-Prep!-

I would say it depends on relative abundace of your target transcript. Low abundant transcripts may require more template to amplify at reasonable Ct.
By the way we routinely use 100 ng of template to reaction and I've seen someone use as much as 300 ng.

-Trof-

oh i see that u have mentioned the Ct value in ur original post.. in that case i think you can increase the template concentration provided you are sure about the well being of other components!!! :)

-Prep!-

ya, im using 100ng too for my starting.....

i faced with the high ct value before too.. and i try to reduce the fold for serial dilution ( means from 5 fold dilution, maybe can change to 4 fold or 3 fold, so the ct value can still in the range..)...

-paramitay-