sequencing result - (May/25/2012 )
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)
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.
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