Help! Unexplained deletion in gene

Ramkumar Lachumanan medp4033 at leonis.nus.sg
Thu Mar 23 23:07:41 EST 1995


Dear Netters,

I am trying to clone and sequence a known toxin gene (180bp) from snake and 
been facing with the problem of deletion (45bp) in the gene . The 
deletion occurs at the same position and there is a frame shift 
where the deletion occurs everytime. 
I'm using specific primers for the gene ( and have tried with a couple 
of primer combination) and the PCR gives a band corresponding to around 
180bp.  
Briefly, the procedure I use are as follows:

Reverse Transcription(RT) using oligo-dT on total RNA
PCR of the RT product using specific primers
Purify amplified band from LMP gel
Ligation into TA vector
Electroporation into JM109 competent cells 
Picking colonies, plasmid mini-prep, identifying clones with insert
Sequencing using M13 reverse/forward primer on ABI automated sequencer

Sincerely looking forward for suggestions. Thanking in advance.

Ram K. Laxman
medp4033 at leonis.nus.sg
National University of Singapore




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