Primers Not working - Real time PCR using SYBR Green (Feb/23/2009 )
I am working on real time PCR using SYBR Green Chemistry. Recently I designed the primers using a software. The results that I got seemed to be perfect. All the design guidelines that should have been ideally met by a primer were met. But when I tried these primers no amplification occurred. I am surprised to see this and cannot understand the reason for this to happen.
I ensured that the primers had Tm within the range 50-60 degrees and appropriate Ta Opt as well. The values of self dimers and cross dimers were also within limits.
Could you please help me with my query ?
Do the primers work in a regular PCR?
have a look at this topic too: http://www.protocol-online.org/forums/inde...?showtopic=6498
i made PCR reaction & RT-pCR, the result was as this :
PCR reaction DNA (sample) give band at 150 bp
PCR reaction DNA(negative control) give band at 150 bp
RT- PCR RNA (sample) give band at 150 bp
RT- PCR RNA (negative control) no band (clear)
** please, what is the Explain of this situation ?
*** this band was primer- dimer or other thing ( as Original band " spesific band" )
thanks for and sorry for elongation