Cloning direction / orientation? (View forum version)


Posted 26 April 2011 - 09:49 AM

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?



Posted 26 April 2011 - 08:19 PM

It works by having sites that will give you different size digestion products depending on the orientation of the insert.


Posted 27 April 2011 - 03:49 AM

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.