Gel purification of plasmid after each of these steps? - (Jul/19/2007 )

Hi, I am planning to do some subcloning as follows:
1) Cut vector with XbaI
2) Blunt ends with Klenow fragment
3) Further cut vector with EcoRI
4) Dephosphorylate vector with CIP
5) Ligate an insert which has one blunt, one EcoRI end
Do I need to do gel purification after each of these steps? Or, for instance, can I add Klenow or CIP straight into restriction mix without prior purification?
Thanks for your help, I have no experience in molecular biology.

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At this point I would remove/inactivate the Klenow fragment. If you have access to a DNA extaction column, you could use that. Or you can use phenol/chloroform.



I would gel purify here. And after gel purification, quantiy the DNA concentration. CIP is a dirty enzyme and over dephosphorylation renders your vector unligatable. So you do need to know the amount of vector you are dephophorylating. It is also a little of a trick to get it right, so some people do not dephosphorylate if the ends of the vector are incompatible (such as in this situation)



At this point, you need to deactivate the CIP. I use phenol.chloroform.



Yes, you can add Klenow into a restriction digest, (along with the appropriate dNTP to fill in the EcoRI 5' overhang). Klenow works in NEB buffer 2 and 3. I perfer buffer 3.

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