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Primers Not working - Real time PCR using SYBR Green (Feb/23/2009 )

Hi,

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 ?

-kate-

Do the primers work in a regular PCR?

-HomeBrew-

have a look at this topic too: http://www.protocol-online.org/forums/inde...?showtopic=6498

-Curtis-

Hi,

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
But
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
saly4ever@hotmail.com

-saly-