Hello,
we are currently working on absolute quantification with SYBR Green qPCR.
We excised from a plasmid containing the cDNA, a fragment, which will be amplified by the primers we used in our assay.
We prepared 19 serial 1:10 dilutions from our fragment, starting with 0.2 ng/µl. However, only the first six 1:10 dilutions show CT differences from ca. 3.3 CT while all the other dilutions have the same CT values.
We are confused because this assay, which was used for the standard curve, resulted in even lower Ct Values when the cDNA from our cells was diluted to test the PCR efficiency of this assay.
If anyone has suggestions we would be happy.
Anne
Did you the your plasmid with a normal PCR to check that the ligation process work?
I always work with the plasmid and when I test a qPCR with the standard, I prepare 8 point of the standard diluted 1:10 (from 10^9 copies to 10^2) prepare in three replicates. And after I run the qPCR-product on the gel to verify the correct product.
Anne Wolf Did you test only the primer in normal PCR? When did you see the qPCR product on gel, you see a single band or not?
Hi,
What Ct values you are getting for your NTC runs? If that is same as your lower dilution Cts then it is the primer dimer.
You can run melt curve to verify its primer dimer or contamination.
Hope this helps.
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