Cloning direction / orientation? - (Apr/26/2011 )
How to check cloning orientation/direction?
People have been telling me to do either sequencing or restriction analysis.
The sequencing machine in my lab is very fully occupied recently, so I can't use it.
Can anyone tell me how to use restriction analysis to check for the direction of cloning?
tell me the protocol and the principle behind it because i don't really understand how this works.
is there any other ways to check it?
It works by having sites that will give you different size digestion products depending on the orientation of the insert.
You can PCR using one primer on your insert and a second primer on the vector. If you get amplification, you know the orientation. You can check the length of the fragment, as well.