checking colonies by vector primers.... - (Jul/12/2006 )
it is very stupid question indeed, but im lost....can we check if the inserted piece is ligated or not by checking it with vector primers??? ..i mean i think no cause how will I tell if the insert is there or no if the primers dont have cDNA sequence....right?
using primers on either side of the MCS will give you a product and inform you whether you have an insert or not and the size of that insert. However, it will not give any information with your insert is specific or the orientation. The best way is to use one vector and one sequence specific primer.
well the idea of kathy seems for me a better approach as screening vectors for presence of insert or not is quite crazy sometimes. and with the methodsuggested, you may not be sure of your PCR conditions.
And as the probability rule that 50% of the positive vectors should have correct orientation, this is not hard to check.
true, one would know if the insert exists or not using the vector primers. If u have the some restriction sites in ur insert, then with proper digests u could find the orientation.
I can only add that although theoritically 50% of the inserts might b of one orientation, I usually found that some sequences have a preference for a particular orientation and the ratios might b 30-70.
The problem with using both primers from the vector insert is one of contamination, not orientation.
Biotechniques. 1998 Apr;24(4):580-2.
Avoiding false positives in colony PCR.
Dallas-Yang Q, Jiang G, Sladek FM.
If you have a lot of colonies to check, the colony PCR with vector specific priers is so quick and easy that it is worth a little unsequrity.
Sequencing primers can be used, because they are always around (at least in labs that have existed a while). If you are cloning a PCR product, of cause you can use the specific primers.