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Repeated mutagensis primer in site-directed mutagenesis


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#1 carylee

carylee

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Posted 07 October 2013 - 09:09 PM

Hi guys,

 

Im trying to create point mutation in domain of gene of interest using enzynomics EZ-MIX kit. I believe this kit follow quick-change site directed mutagenesis method; PCR amplification of forward-reserve complemantary primer with the introduction of mutation in the middle of primer, follow by dpn1 digestion.

 

I am able to get the colonies after transformation but when i sent two clones for sequencing, sequencing result turn outthere is point mutation, but the whole primer region duplicated in both clones. May i know if anyone had experience this and how to overcome this problem. Im thinking sending more clones for sequencing may help.Thanks!!

 





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