Adapted from a protocol I got from Kelly Bode
Note: If you use the custum primer version of T3/T7 you have to reprecipitate the DNA to get rid of the NH
salts as they inhibit the reaction.
T3 or T7 primer (10 pmoles or 100 ng)
5X Forward Rxn Buffer (Gibco-BRL)
T4 Polynucleotide Kinase (Gibco BRL) (5 U/ul)
P]ATP Amersham -20° (clear)
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.