What is going on with this qPCR assay?

[Buni]

Hi all,

I have attached an amplification plot from a qPCR assay I am trying to optimise. The efficiency of the reaction is fine (99%) however the curve just does not have a nice sigmoid shape. The standard curve is generated using plasmid DNA however the assay fails to amplify positive control genomic DNA.

It has been suggested to me that I need to perform a temperature titration as the annealing temperature may be too high. As far as I know the qPCR machine that I use does not have the ability to perform thermal gradients. Knowing this is it worthwhile for me to perform the temperature titration using the same primers in a conventional PCR assay?

Any ideas/tips/feedback on what you may think is going on with this assay is greatly appreciated.

Cheers

[thehulk]

It looks like you're using the Stratagene Mx which does not allow you to use any sort of thermal gradient. But what is your highest copy number of your dilution series? To me it looks like the reason you don't see the textbook sigmoid-shaped curve is because your dilution series isn't starting early enough and they all get cut off at 40 cycles. Try extending to 45 cycles. Also, your replicates look very messy. What master mix are you using? In addition, you may not be able to amplify gDNA if your assay is designed over an exon boundary.