DNA contamination in RT-PCR

Isidre Gibert igibert at cc.uab.es
Fri Oct 27 13:01:10 EST 1995


       I am working with the RNA of Rhodobacter sphaeroides.I am 
carrying out RT-PCR to determine whether a certain transcript exists or 
not and Northern experiments have been unsuccessful.
       My problem is that I have DNA contamination in the RNA 
extractions, and the band is amplified even if RT is not present in the 
reaction mix.  
      I have tried several extraction protocols: phenol acid extraction, 
TriPure reagent (from Boehringer Mannheim) and a method described in 
EMBOJ (93) 12:3957-65. In all cases I have tried different initial 
quantities of cells ( as little as 15 µl exponential culture) but, the 
band is always amplified. Following DNAse treatments don't improve the 
results.

Thanks,		
										Isidre





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