Quantitating Radioactive label in DNA and RNA

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Sun Jul 3 19:32:42 EST 1994


>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
>> advance
>>                 Heidi Hinkel
>>                 hlhinkel at matt.ksu.ksu.edu
>>                 Division of Biology
>>                 Kansas State University
>>                 Manhattan, KS 66506
>
>Heidi,
>
>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.
>
>Materials:
>
>Acid solution:keep ice cold
>1.0 M HCl
>1.0% NaH2PO4
>1.0% Na4P2PO4 (Sodium pyrophosate)
>
>95% Ethanol (cold)
>
>Whatman GF/B Glass Microfibre filters (2.4 cm; Cat# 1821024)
>
>Procedure:
>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
>the filter.
>
>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
>minutes).
>
>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.


There is a much quicker, simpler method of estimating incorporated from
unicorporated nucleotide using a TLC method with a PEI plate that I posted
many months ago if anyone is interested.  The whole thing will take less
than an hour (including counting).

Cheers, Klaus

*************************************************************************
Dr Klaus Matthaei                               |           |
The John Curtin School of Medical Research      |  _--_|\   |
The Australian National University              | /      \  |
Snail mail: Canberra, ACT 0200, Australia       | \_.--._/<<|
E-mail: Klaus.Matthaei at anu.edu.au               |       v   |
Landline: 61 6 249 3782
                        "Sometimes I'm the Louisville slugger"
                        "Sometimes I'm the Ball"
                                Dire Straits
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