Cloning help - how to break down a restriction site! (Apr/11/2007 )

Hi there,
I want to eliminate a restriction site form my cloning vector. This is my plan, but I need confirmations, suggestions and tricks....
I have to digest my vector with NcoI, remove 5' protruding ends with S1 nuclease and self-ligate the vector.
Suggestions are welcome on optimisation of all the abovementioned steps, but the main question is....how to improve self-ligation of blunt end digested vector versus self-ligation of vector still containing 5'-protruding ends (not or poor digested with S1 nuclease)?
All suggestions are really welcome.

After the ligation reaction in your proposed strategy, I would suggetion a second NcoI digest. This kill digest would destroy any religated NcoI molecules that have not been blunted by S1 nuclease.

In addition, adding PEG 6000 to a final concentration of 10% into the ligation buffer will help with blunt end ligation. You should also ligate the plasmid at dilute concentration. When the plasmid is at a lower concentration, it tends to religate to itself.

But bewared, if DNA is too dilute it will not ethanol percipitate. Use dextran to help percipitate the DNA.

Also note that you must clean your DNA, after the digest (ethanol percipitate and resuspend in sterile distillled water), some buffers contains detergent and will hamper transformation efficiency. PEG also reduces transformation efficiency. Ethanol percipitation will remove PEG as it is soluble in ethanol.

Excellent suggestion to re-digest with NotI after ligation!
thank you very much

I have to chew up 5'protruding ends hence I must use S1 or mung bena nuclease...

Does anyone know the right temperature to do Mung bean nuclease reaction? Some prtocols suggest 30° C some other 37°C!!!
A little bit confusing....