If you are cloning a gene into expression or reporter vector, it is better to confirm the presence of insert by using vector-specific primers. Sometimes, nonspecific nuclease may chew up sequences around the cloning site, and that leads to no expression (even the insert is relatively intact).
In general, it is wise to screen colonies with vector-specific primers in the first round. Then after streaking for single colony, screen a second time with one primer based on vector and the other based on insert sequence.
Sequening primer is generally too short for PCR. You need to make a longer primer (20-21mer) using more bases around the site.
Last edited by relaxin
on Jul 30 2012 11:57 am, edited 1 time in total.
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