Help! Unexplained deletion in gene

Robert Brushia ez017144 at peseta.ucdavis.edu
Mon Apr 3 19:45:29 EST 1995



I don't really have an explanation for your problem but I would suggest 
checking your RNA, perhaps it has the deletion already in it.  One way 
you might find the step at which the deletion is occuring is to use one 
primer that is complementary to the deleted region for your PCR:  when 
you stop getting a product you will know at which step the deletion is 
occuring.  If you can't get a product from the original cDNA then the 
45 bp of sequence may have been deleted by reverse transcriptase.       

Rob Brushia			Dep't. of Med. Biol. Chem.
				School of Medicine
				UC Davis
				Davis, CA 95616
				(916)752-6067

                                                                       
 On 24 Mar 1995, Ramkumar Lachumanan wrote:

> 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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