real time PCR using two sets of primers with different melt curves - (May/29/2007 )
I would really like some advice, I am performing real time PCR using sybr green for detection of two separate products using primers with different melt curves. The two products are SNP variants and the PCR is performed after chromatin Immunoprecipitation. After I designed the primers I standardized them using plasmids containing the SNPs and they worked beautifully, good amplification and distinct melt curves. When I tried using the same in my ChIP samples (not multiplexing, separate tubes) ,I did not get amplification at all!!! Would love some feedback, this are the last few experiments I need to get done before I wind up my PhD thesis, so please help!!!
Which size should your amplicon be? After Chip, you are more likely to amplify very short sequences.
The product size is around 190 bp.
Okay, size is suitable. The startung amount of DNA after CHIP is much lower than your plasmid DNA so I'd guess you need more cycles or a (hemi-)nested strategy. Optimization with regards to primer and template concentration may also differ between plasmid DNA and CHIP derived DNA.
sorry for the delay in adding on to my own post. Could you give me more details on hemi nested strategy? or a website that has info