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sequencing result


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#1 priyamvada

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Posted 25 May 2012 - 11:52 AM

I cloned a gene recently (~1kb) and got it sequenced. after that i compared my sequencing result with the gene sequence reported in EMBL. However they don't match!! but wen i take the REVERSE COMPLEMET of my sequence result, the two sequences match perfectly. what do i make out of this? (im new at cloning)

#2 neuroelectrophysiologist

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Posted 25 May 2012 - 02:12 PM

Are you cloning into a blunt end or a single sticky end? Your insert could therefore ligate in either orientation. It is a 50-50 chance whether your insert ligated in the correct orientation.

You should pick more colonies and send a few more away for sequencing.

#3 priyamvada

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Posted 25 May 2012 - 04:46 PM

the plasmid sent for sequencing was pGEMT-easy cloned into DH5alpha. so i dunno if the concept of blunt/sticky ends applies here cuz it was cloned based on the A-overhangs. However i have Bam and Xho sites in my primers so that i can later on ligate my insert into an experssion vector




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