3'DNA biotinylation kit - (Jun/21/2004 )

Hey all,

Have been biotinylating DNA using Pierce's 3'DNA end labelling kit. However, I can't detect anything by ECL, not even control samples when i am doing a blot to estiamte labelling efficiency.


Am immobilising DNA to HYbond N membrane

Using 3% BSA in PBS to block membrane, rinsing in PBS Tween and probing with HRP-STreptavidin conjugate (1:500), but not detecting anything by ECl.

Anyone having any similar problems? Anyone have any advice?

Much appreciated

Shaunyb

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Hi,

we also have a problem with EMSA...

We use endlabelled biotinylated dsDNA (labelled with Pierce-Kit) an want to do EMSA with the Pierce lightShift-Kit...

There are no problem with the gel, our "simple" problem is to DETECT the biotin-dsOligos (some 30mers) on the gel, we can detect the non-labeled DNA but not the biotin-tagged version. The results are the same with the positive control of Pierce?

We also can´t see anything on membranes!!

We can´t move on without solving the problem!!

Do you have a helpful hint?? Could you solve the problem??

Thank you very much in advance!

Best wishes,

Gerhard

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Just a couple of questions:

what sort of gel do you use and how do you transfer? If you use agarose, I am all over that one...I do it all the time and it usually works pretty well for me. If you use PAGE I don't have any hints for you.

A

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