In-fusion advantage PCR cloning kit - --- failure after many many months-- (Aug/12/2009 )

Great to hear of your success! It's always good to see progress... even if it isn't in your own project!!

Next week, the PCR linearisation begins!

Have a great weekend folks!

swanny on Aug 21 2009, 04:09 PM said:

Just to let you know- the sequence came back pretty good. I sequenced 3 of each vector and out of each of them there was only one which had an additional base at the site of ligation.

Hope your PCR linearisation is going well. Thanks for all the help/suggestions.

Cheers, Teagan

I've been working with the infusion kit for a few months now and had great success. We've found that performing several double digests on our vector over a few days cut our background down to maybe 1% (18 colonies). I digested my vector with AfeI/XhoI for 5 hours at a time and then gel purified it. I then performed another double digest the next day and then phenol/chloroform extracted and precipitated it. I repeated this 1 more time (3x's digested in total). I checked for background with vector + ligase and got 18 colonies. I made sure to gel purify my PCR insert. Then I used the infusion website to calculate amounts to use in my infusion rxn (2:1 ratio) and diluted the completed reaction with 40 µL of 1X TE buffer. I then combined 1.5 µL of this dilution with 50 µL of DH5alpha competent cells and plated about half on one amp plate and the remaining half on another amp plate. Plate one yielded 1000 colonies while plate 2 yielded 769 colonies. We submitted DNA for sequencing which confirmed the presence of our insert. Hope this helps with any future projects.

On the subject of removing background from religation of a single-cut vector for SLIC or In-Fusion cloning, has anyone tried using a phosphatase (CIP or SAP) to prevent re-circularization of the vector? None of the protocols seem to mention it, and I can't figure out why. Is the assumption that, in the absence of ligase, re-circularization is irrelevant and that the background must be uncut DNA?

Anyone got any insight? I haven't tried it yet, but it seems like it might help.