Simoltanious Multi-restriction digestion
Posted 18 June 2012 - 09:41 PM
I need to cut out an insert from a plasmid with XhoI and ApaI. its a rather long fragment(7.5 kb). in order to not purify the digested fragment out of agaros gel, I am going to digest the vector fragment further to inhibit it from taking part in the finla ligation reaction. I can use EcoRI since this enzyme dose not digest the Insert. then I am going to clan this reaction up using a column based kit( roch) which unfortunately wont pass vectors digested fragments through. thats why I would end up with 3 fragments: 1- insert 2, 3 vector. is using these fragments in the ligation reaction with pcdna as the destination vector going to reduce ligation efficiency beyound repair?
any suggestion is grately appreciated.
Posted 19 June 2012 - 03:23 AM
Posted 22 June 2012 - 05:28 AM
Posted 22 June 2012 - 06:33 AM