HELP please: Inverse PCR with genomic fragments!

user user at garfield.neuro.ruhr-uni-bochum.de
Wed Feb 7 11:47:39 EST 1996


Dear netters,
the following problem is driving me crazy: I try to circularize pools of 
genomic DNA-restricition-nuclease-fragments of distinct sizes (2-4 kb). Among 
these circular molecules there should be one that contains a certain gene 
whose promoter region I want to clone. So far, I have tried to manage it by 
inverse PCR with primers derived from the known protein-coding region. But 
after 3x30 cycles there is no product! Another PCR-based method to catch 
promoter regions is the Rapid amplification of genomic ends (RAGE). No 
success, too. 
Who has already solved the problems of a) effective circularization of 
genomic DNA, b) suitable conditions for RAGE-ligations and RAGEs, and c) 
inverse PCR with 0,001% target molecules among many others ?

Thanks for any answer!

Claus Graßmann, Department of Biochemistry, Ruhr-Uni Bochum




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