Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

primers fpr sequencing - (Mar/30/2009 )

Hi everyone
I had have inserts in some vector like pMW172, pcDNA3 and pcDNA3 FLAG, and I want before starting gene expression to check the inserts by sending them for sequencing so I need a help to find forward and reverse primers for these vector/ inserts.

Thank you in advance


look at the plasmid map, look for names like M13 forward or sp6/t7 or bovine growth hormone A (BGHA)


you can even design a primer of your own for sequencing based on the map!


pcDNA has a T7 sequencing primer.
If I remember correctly, pMW172's back bone is pET. If I'm right, then it to has a T7 sequencing primer.


it also depends on the size of your insert, if it is a bit large, say >1.5kb, you better design two more (one forward, one reverse, 18~21bp each) which will anneal to your insert.