REQ: PCR primers - help wanted

[9605574b at udcf.gla.ac.uk]

To whoever may be of help,

I am currrently using a set of six primers in order to make deletions in the 
gene for a human enzyme. All but one of my primers is working using the same 
conditions. The one which isn't working is a G+C rich (66%) 30mer with a 
melting temperature of 75.7 degrees C. There is a 12 degree difference in 
melting temp. between this one and one of its pairs. Is this of any 
significance?

I have tried varying the denaturation times and the extension times. My 
annealing temperature is 50 C which may be slightly low. There is also the 
possibility that my primer is rather large and may have a secondary structure, 
however I do not see any bands which may indicate primer dimerisation.

Thanx


FAB  ;->