is there any trouble that would result from incubating Quick CIP for 1 hour. NEB recommendation is to do it for 10 min only, isn't that a bit too short or has anyone tried it and it works just fine.
Taking in consideration that I will be adding it to the reaction mixture after performing double digest (50 mcL reaction)?
Check https://international.neb.com/products/m0525-quick-cip
https://www.med.upenn.edu/robertsonlab/assets/user-content/documents/Preparation%20of%20Vectors%20Treated%20with%20Alkaline%20Phosphatase.pdf
Thank you so much Dr. Chinaza, I checked the neb recommendations and they recommend incubating thr reaction at 37 C for 10 min I just wanted to see if it is possible to incubate it longer to ensure the reduction of the background when I do the ligation step followed by transformation
I tried using CIP during enzyme digestion before ligation (1 hour - 37 C) and it seems that it is not working, my plasmid is recirculating (lot of background in control condition).
Hi Hugo
I actually incubated CIP for 1 hour at 37C and it worked well for me, may be you can check that the enzyme is still working by using a control
Also make sure to use CIP on the plasmid only not the insert. Recirculation of plasmid may occur but it should be in very low percentage especially when doing double digestion
Best of luck
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