Primer extension analysis

Morten Lukacs Morten.Lukacs at veths.no
Mon Apr 21 03:38:50 EST 1997


Hi netters,

I'm trying to map the 5' terminal of mRNA by primer extension. I'm using 

radiolabeled primers and nucleotides. But the problem is with the analysis
on denaturing polyacrylamide (7M urea) gels.

The labeled product migrates with the same size as the "top" of my 

MW-markers (sequence ladder). This happens whether I run the gel for 2 or 6
hours. 
The products is expected to be 150 and 300 bp, but these products also 

migrates as the same size!

I have tried different: % polyacrylamide, temperature of denaturation of
products.....

But nothing works....so whats wrong.....?
Should I use formamid in the gels....?
Is my products just nonsense????

OK, so any help will work for me!!

Thanks

Morten


Morten F.Lukacs
Norwegian College of Veterinary Medicine
Dept. of Biochemistry, Physiology and Nutrition
Postbox 8146 Dep., 0033 Oslo
Norway




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