Note: If you use the custum primer version of T3/T7 you have to reprecipitate
the DNA to get rid of the NH4+ salts as they inhibit the
reaction.
Mix together:
T3 or T7 primer (10 pmoles or 100 ng)
X µl
5X Forward Rxn Buffer (Gibco-BRL)
5.0 µl
T4 Polynucleotide Kinase (Gibco BRL) (5 U/ul)
2.0 µl
[gamma-32P]ATP Amersham -20° (clear)
5.0 µl
water
Y µl
50.0 µl
Incubate at 37° for 10 minutes (Note - Kelly says that times
greater than 10 minutes are only useful for getting no results). Stop
the rxn with 1 µl of 0.5 M EDTA.
Meanwhile, you have been prehybing your filter. Add the probe directly to
7 ml of hyb solution. Incubate for at least 2 hours at 42°.
Wash 2 times for 7 minutes each with 2X SSPE, 0.2% SDS at room
temperature.
Put on film overnight. There will be a certain amount of background.
This page is, as always, Copyright (c) 1997 by Craig D. Amundsen. All rights
reserved.