Troubleshoot ARMS PCR - (Mar/08/2009 )

Dear All,

I'm using ARMS PCR method to detect common mutations in beta thalassaemia samples. PCR product for certain samples seems to be fine but having difficulty in amplifying some samples for the same mutation analysis. Can anyone advise me pls. thank you.

Mag

What does it mean having dificulty amplifing some sequences? Not a single product or missing the allele specific ones?
Do you use similar DNA concentration? Is your DNA a good quality? You can do some check on a different gene, like actin for example.
If everything seems fine, and you miss the products there may be some sort of deletion in the area of your primers, especially when you look at thalassaemia pacients.

Hi I am also using ARMS-PCR technique to detect mutation at a known site of a cytokine gene. I have done hundreds of samples on my already optimised protocol but now I am in trouble. Already optimized protocol only amplify internal control gene with very weak cytokine gene bands. I am unable to read theses weak band. Could you please help me in this regard?