In article <2us9ns$eso at matt.ksu.ksu.edu>
hlhinkel at ksu.ksu.edu (Heidi Lanora Hinkel) writes:
> I am looking for methods with which to quanitate the
> amount of P32 -ATP incorperated by end-labeling with T4 kinase,
> in both DNA and RNA probes. I would like to hear of any methods
> that you know of and which one you think is best. Thanks in
> Heidi Hinkel
>hlhinkel at matt.ksu.ksu.edu> Division of Biology
> Kansas State University
> Manhattan, KS 66506
We usually quantitate incorportation by measuring acid precipitable
counts on a glass filter. Here is the procedure:
First purify your probe by gel filtration. Use your purified probe in
the following proceedure.
Acid solution:keep ice cold
1.0 M HCl
1.0% Na4P2PO4 (Sodium pyrophosate)
95% Ethanol (cold)
Whatman GF/B Glass Microfibre filters (2.4 cm; Cat# 1821024)
1. Add 1.0 ul of your sample to 10 ug carrier DNA (final vol 11 ul) in
an eppendorf tube
2. Add 1.0 ml of the cold acid solution to the DNA, incubate on ice
for 15 minutes.
3. Place the glass filter over a vacuum manifold (milipore XX2702550
or reasonable substitute).
4 Draw the vacuum and add 5 mls of the acid solution to equilibrate
5. Stop the vacuum and add the sample suspension.
6. Draw the vacuum. When the sample has been drawn through add 10 mls
of the acid solution to wash.
7. Add 5-10 mls of cold 95% EtOH, vacuum to near dryness (2-3
8. Remove the filter from the unit and place it into an LSC vial. Add
5.0 mls of LSC cocktail (we use biofluor) and count. Compare to 1.0 ul
of your probe alone in LSC coctail.