Is it pointless to check orientation if non-compatible ends are used? - (Aug/29/2009 )
If I cut my vector and insert with AgeI and NdeI which produce ends which are not compatible with each other, (and then ligate them) is it suffcient to just check that my insert is present in the vector, without checking orientation? I should think it is but my cloning-fu is still weak.
seanspotatobusiness on Aug 30 2009, 05:06 AM said:
I think it is suffcient. However in my lab. we usually sequence one or two clones after they pass the pcr verification so we can know if there are some mutations or not.
I agree with clwop -- you're likely going to need to sequence your cloned insert anyway, and this data will confirm orientation (though I also agree that a true clone will very likely have the insert in the expected orientation).
How are you screening your transformants for the presence of an cloned insert? If you do it by PCR using a primer that binds to the vector and one that binds to the insert, you can detect positive clones and confirm orientation in one step.