Hello all,

I have been trying to quantify a 292 bp in my community DNA. In order to quantify it, I am using absolute quantification approach. I amplified the gene (292bp), purified it (ratio 260/280 is 1.7 to 1.85, gel image shows sharp desired band), followed by its standard dilution (varying 1:2, 1:10, 1:5). I am using Quantitect RT-PCR Sybr based kit. The machine is 7500 ABS.

I always get a R2 value of greater then 0.995, but the efficiency is 60 to 70 per.

Primer concentration is 200nm. Mgcl2 concentration provided in kit mix is 2.5 mM.

So, I decided to increase the Mgcl2 to concentration to 4mM and primer to 300nm to 400nm. The efficiency is increased to 75 to 80 per.

Now after so much of trial, what could be the probable factors that I am not achieving good pcr efficiency and slope.

Is it because of kit components such polymerase?

Is it because of machine calibration issues?

Are there any other factors that can be improved?

Please suggest.

Thanks.