southern (discomfort)

Vivian Miao vmiao at unixg.ubc.ca
Tue Jan 23 22:39:05 EST 1996


In article <4e2u0g$get at eldborg.rhi.hi.is>, php at rhi.hi.is (Petur Henry
Petersen) wrote:

> I use depurination , denaturaring,neutralisising steps and then 20* SSC.
> I would like to know if the time of these steps is in any sense crucial. 
> I use EtBr to measuere DNA left in gel and it seems to me that a whole lot
> is left behind. How important is it hthat the pH of the neutral-buffer is
> 7.5 ? Is it possible to blot too long ( in the SSC)?

> ps: why is it neccesary to neutrailse the gel? why not blot the denaturared
> product and then neutralizise the nylon film (that is what I am using
>  (Hybond N).

For getting all the DNA transferred, have you considered blotting with 0.4
N NaOH after depurination?  For one thing, you don't have to pH the
solution, and 16 g NaOH pellets /liter is pretty speedy to make!  I
depurinate for 15 to 20 minutes, and blot from 4h to overnight and restain
the gel to check for completeness of transfer.



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