T3/T7 Mapping
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 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.