Problem with absolute quatification - absolute quantification (Jun/29/2009 )

Hello,

We have recently got a ABI 7500 real time PCR. We do absolute quantification of virus cDNA using Plasmids as standards.

Every time we have to make a new batch of standards we run them without dilution to estimate what is the Ct value available for the batch. The SYBR mix used for the real time is from Qiagen QuantiTect SYBR green PCR master mix Catlog No. 204145. It comes with a passive dye ROX.

But after the plasmid prep the Ct values we got were very high, Ct of 27 etc. The melting curve was good and there was no secondary peaks seen. When we ran the gel the bands were so thick and bright that we were doubtful why the Ct showed 27.

So we set up a reaction using the plasmids (4 batches) and made a single master mix and ran the same reaction in two different machines, The first one was ABI 7500 and the other Biorad. The Ct's we got from Biorad was 7 were as the ones for ABI was 27.

I have attached the excel sheet with Ct values from ABI and MJ. I am not sure what went wrong.

Could anyone suggest any ideas?????



thekid

Hi,

I don't know whats going on with it, but did you run your samples in triplicates? Also your Tm value for NTC is very high on the biorad machine Vs ABI, so that suggests that there are different products. I hope you solve your issue.

Thanks,
-Uma