Posted 29 January 2011 - 02:11 PM
I'm not sure if I understand the principle of the "Topo Cloning" correctly, so I hope that
some can tell me if I'm right or wrong:
- the vector is linear and is looking somewhat like this
5' ------------------T-P-Topo 3'
Here there is the topoisomerase enzyme covalently bound to a phosphate of a thymidin resudue at the
Question: Are there also a free phosphate residues on the 5' ends?
- because a free 3'-OH end is missing the vector can't ligate itself to become circular
- if I add my PCR-insert with the free 3'OH ends the topoisomerase will use these OH groups
to connect vector and insert
Question: If the 3'-P-Topo end of my vector connects to the 5'-P end of the insert what will
happen to the second phosphate?
I could imagine that it could be used to connect the 5' phosphate free end of the
vector to the 3' of the insert!?
I hope someone can give me some good explanations on how that method exactly works
Posted 31 January 2011 - 12:19 AM
Posted 31 January 2011 - 02:24 PM
Posted 01 February 2011 - 05:42 AM
Ok, you think that there are phosphates on both, phsophates on the 3' ends of the vector and also at the 5' ends of the vector DNA?
Posted 01 February 2011 - 02:36 PM