alkali vs non-alkali transfers

Victor Levenson levenson at uic.edu
Fri Jul 18 15:35:56 EST 1997


Ross S Feldberg <rfeldber at emerald.tufts.edu> wrote:

>Hi;

> I am setting up Northern blots and was wondering about optimal transfer
>conditions. We are using downward blotting onto charged nylon filters with
>an alkaline transfer solution. The protocol we are following calls for
>only a 60 min transfer in order to minimize hydrolysis of the RNA.
>However, I am not sure if we are getting good transfer in this time frame. 

>I was wondering if anyone uses non-alkaline transfer medium or whether
>hydrolyis is enough of a problem to worry about. How long do folks
>generally transfer for?  (I ask because our initial efforts with an actin
>probe gave a pretty weak signal).


>Thanks

>Ross Feldberg
>Biology
>Tufts Univ.
>rfeldber at Tufts.edu

Ross,

It all depends on the concentration of NaOH; if you use 50 mM at r.t.
the transfer (upward) can go for up to 8 hr. I used 6 hr with charged
nylon membranes and it worked much better that neutral salt buffers.
My colleague was so impressed that she changed o/n salt to 6 hr
alkaline and then even shortened the time to 5 hr with good results. I
think that 60 min is just too short a time to get good transfer.

Hope that helps,


Victor Levenson
Res Asst Prof
UIC
levenson at uic.edu




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