Stability of a transgene and its expression - (Feb/17/2007 )

Hi

I am part of a group working on detection of transgenics. Now a day, I am facing a problem regarding detection of GM cotton having insecticidal transgene. However there are no positive amplification using these samples but positive signals measured using ELISA, although there is very low expression. For PCR detection I have been used 5 different primer sets, but no amplification observed.

So now my results are very contradicting, ELISA showed but PCR not. DNA is of good quality and has been checked for the amplification of marker gene (npt-II) and internal control gene (SadI).

What may be the possible reason..............?

Please guide me.

Thanks and regards

chandra

Maybe you just haven't found the best amplification conditions for your PCR yet. For example, if it is a GC-rich gene, you may require DMSO or betaine to amplify your target.

Thank you Sir for kind reply.
But I have been tried addtion of DMSO, touch-up, touch-down PCR, hotstart etc and no amplification observed. Even gradient PCR and gradient of templated DNA (from 10ng to 1ug) was tried but no amplification observed. while expression of protein was observed thru ELISA.

So still question is same.

Thanks and regards

chandra

I'm not an ELISA expert at all, but perhaps something else is causing the positive ELISA result? Is it possible to do a Western blot to check?