Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Help with cloning!. - Cloning problems (May/10/2009 )

Pages: Previous 1 2 3 

micky74 on May 12 2009, 03:01 PM said:

So you do not check for the vector/insert ratio? Or you check by visual inspection on a gel? How many ng of vectors more or less for each ligation?
At the end you run also vector alone and vector= insert and you alway obtain more colonies on the vector=insert or you do not bother to check that?

I very rarely check for ratios, but I usually have a rough idea how much insert I have by the intensity of the band produced by the PCR. Ditto for the vector. We no longer bother to do vector alone controls. We don't even own a spec, though there's a nanodrop in a neighboring lab, should the need arise.

As I've stated before in other threads (and I know it's heresy around here), such things (in our hands, anyway) are not critical -- cloning in the lab has become routine -- we usually get what we're looking for on the first or second try. I'm not saying there's never a difficult clone, just that it's the exception, not the rule.

The only things we do that are perhaps different than other labs is to use guanosine in TAE to cast and run gels from which we're going to recover fragments (to protect the DNA from UV damage), and use the glassified Ready-to-Go ligase linked to above.

Pages: Previous 1 2 3