PCR primer problem

David Fogarty onxfoxxd at lgdx02
Wed Nov 15 12:35:24 EST 1995


Loredana,
It seems to me that your annealing temperature is too low.  Thus your 
3 prime primer may be sticking to many non target sequences and be being 
used up in non-specific transcript generation.  What concentration of 
primers are you using?  Try using 1 or 1.5mM Magnesium.  Raise your 
annealing temp to 60 degrees.  

I say all this because recently I have been working with a set of primers 
which I designed to amplify receptor transcripts.  Initially I used what 
I thought were low stringent conditions ie. low annealing temp, high 
Magnesium concentration and high primer concentration.  Bands were weakly 
amplified.  Now I have switched to more highly stringent conditions, with 
0.1 microMolar primers, 1 mM Magnesium and 60 degree annealing with 
"touchdown" to 56 degree.  Results have been spectacularly improved and 
bands are now clearly present or absent in EtBr gels.

Why not give it a go?

Good luck,

David J.  Fogarty
Department of Neuroscience,
University of the Basque Country (UPV/EHU)
Bilbao, Vizcaya,
SPAIN.



More information about the Methods mailing list