CIAP dilution - (Nov/02/2012 )

I have a vector which is double digested with NotI and BamHI and is about 5.4kb long hwich needs to be trated with CIAP.
I am nit sure how much to dilute my CIAP and how many microlitres i need to use in the ciap raection.
what would be the best incubation time and at what temperature.
I see from protocol at 37C for 15 min and again 56c for another 15 min which is then added with another aliquot of dilluted CIAP and repeat the incubation again.

there is something like this in the protocol about dilution and i do not really understand.Conc of the CIAP is 1u/ul


Dilute sufficient CIAP for immediate use in CIAP 1X Reaction Buffer to a final
concentration of 0.01u/µl. Each picomole of DNA ends will require 0.01u CIAP.
(1µg of 1,000bp DNA = 1.52pmol DNA = 3.03pmol of ends.)


Please help me out in the issue so tht i can proceed with my ligation stuff
and how to check if the CIAP worked????

Thanx in advance

Harry

First off - as Not1 and BamH1 are not compatible ends you shouldn't need to phosphatase treat your vector to prevent self ligation.

However, for the dilution - you need to know how much DNA you have in your reaction, from that you can calculate the pmol ends (the number of ends on your DNA in picomol). A calculation tool can be found here. You can then mulitply this number by 0.01 to get the amount in units you need to add, this will then correspond to the number of ul (1U/ul).

1 Unit means 1 microMol Substrate in 60 minutes processing by the Enzyme (Enzyme Depending).

Think homeopathy. I'd recommend a dilution of CIAP of, say, 10,000,000,000,000 to one. Translation: I'd never use it. At all. Makes more problems than it solves, in my opinion.

thank you all for the answers

Listen to phage. It is a pain in the ass. If it is not completely inactivated it will make your life hell during downstream procedures. It can cause some funky effects on linear plasmids.