I am curious whether I can short cut the protocol by directly using the antartic phosphatase (NEB) treated plasmid to the ligation without prior purification to remove salt/protein? Does anyone deal with this before? Will it interfere the ligation reaction?
The next thing it will dephosphorylate is your insert! So of course it will interfere.
Hi Tassanee,
if you have heat inactivated it then you should be OK to use it without further purification - it's even stated on NEB's web page.
Now if you have double digested your plasmid, I would recommend purification of the vector as otherwise you will still have the short fragment from the MCS which will compete for the free ends with your insert. Whilst these won't be ligated they will reduce / stop the ligation of your insert.
My view:
For the sake of a quick purification, you could be saving a lot of time down stream by purifying the vector. So I'd recommend always purifying the vector, even if not absolutely required.
-Richard
https://www.neb.com/products/m0289-antarctic-phosphatase
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