Digest for insert orientation - (Jul/22/2012 )
I digetsed my plasmid (pGreen) and my inserts with the Xho and Sal enzymes and ligated them together before transforming e.coli. The insert appears to be present in the plasmid however I now need to check the orientation of the insert as Xho and Sal have the same 'sticky ends' and my cloning is therefore not directional.
Is it possible to check the orientation of my insert using a restriction enzyme digestion with these enzymes? Or should I just sequence it?
I think I've worked this one out for myself. By digesting with either Xho or Sal and digesting with an enzyme which cuts within my insert, I can work out which way it has gone in depending on the sizes I get from the digestion
Correct. You should still sequence the insert if you PCR generated it, just to make sure there were no errors generated.
you can also perform colony PCR using vector-insert primers to confirm correct orientation.
One of the directions of insertion will not cut with either enzyme. The other orientation will cut with both.