Blunt end cloning - (Jun/19/2014 )

Cheers for the hot tips :) Primers should arrive on Monday.

Bio-Lad on Fri Jun 20 12:33:52 2014 said:

I agree that PCR amplifying the promoter may be the easiest way to go about this but while you are waiting for your primers to arrive, you may want to try one other thing first.  Since you (hopefully) still have the fragments you digested out, I would suggest trying the blunting with Klenow rather than MBN.  I've tried doing similar ligations before and had quite a bit of trouble getting the MBN to actually do what it was supposed to do and ended up with no colonies as well.  I repeated using Klenow instead and it worked fine the first time around.  Admittedly, it could be something wrong with the way I set up the blunting reactions (can't omit human error) but I've never gotten very good results with it.

Thanks again! Really appreciate the help guys :D

phage434 on Fri Jun 20 13:19:45 2014 said:

I agree with Bio Lad, that Klenow would be a much better choice than Mung Bean nuclease. Also, avoid CIP in favor of shrimp alkaline phosphatase or antarctic phosphatase (and use minimal amounts of these, as well).

Hi!

Just wanted to thank you for all your suggestions :D I have clones! wOOt!

It seems like we have a contamination in the lab though which is making everyone's cloning a pain in the behind. I'll start a new thread about that.

Thanks again!

Clare