qPCR assay optimisation tips

[Buni]

Hi,

I am trying to optimise a qPCR assay to establish a standard curve suitable for use in quantification. I am currently using a dilution series of plasmid DNA to establish the standard curve (see attached). The assay is efficient (99%) however there is not a nice sigmoid shape and a positive control of genomic DNA is not being amplified.

It has been suggested to me that I should perform a temperature titration as the current annealing temperature (60°C) may be too high (the primer set annealing temperature is 50°C). As far as I know the qPCR machine that I use does not have the ability to perform thermal gradients. Is it worthwhile for me to perform the titration using the same primers in a conventional assay?

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

Cheers

[namishu95]

You may try a gradient assay on conventional thermal, but make sure the ramping is similiar, also try to modify yout primer.

[MEDIBENA]

did you linearized the plasmid?

[wilczka]

Hey,
If you make your gradient in a conventional thermal you should use your supermix fron realtime reaction. Recently I've experienced how supermix may inhibit the reaction espacially when compared to conventional one.