3 replies to this topic

[mahrak]

good day all

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.

[phage434]

This should work fine.  There will be some efficiency loss, but there is in most more standard reactions as well.

[mahrak]

tnx a lot phage. any suggestions on performing the digestions? seqencial? how about adding all three enzymes together? do they work if add them sequencialy to the very same reaction?

[phage434]

You can do the digestion with both enzymes in NEB buffer 4.  ApaI works at 25C, so you should digest for 1/2 hour at 25C (room temp) and then digest an additional 1/2 hour at 37 for the XhoI by moving the tube.  Your PCR product should digest without difficulty, assuming you have extra 5' bases on the PCR primers.  The vector may not digest well, due to supercoiling and possibly due to overlapping dcm and dam methylation.  You might want to check vector sequence to see if there are methylation sites.  You might need to add extra enzyme for the vector digestion.  Heat kill the enzyme and ligate without purification.  Don't be tempted to make the majority of the volume your DNA solution.  This often causes RE inhibition due to impurities.  Better is to dilute the DNA in the RE reaction.  You don't need much DNA in the final ligation.