Protocol
5'-[32P]-Labeling of oligodeoxynucleotides
- List the following reagents and incubate for 30 min at
37°.
|
reagent |
amount |
volume |
Final concentration |
T4
polynucleotide kinase*
10 units/mL |
10
unit** |
1
mL |
0.2 unit/mL |
|
Oligodeoxynucleotide*** |
50-300 pmol |
1-39 mL |
1 to 6 mM |
| [g-32P]ATP (6000 Ci/mmol) |
50 mCi, 8 pmol |
5 mL |
0.16 mM |
| 10 X Buffer* |
|
5 mL |
70 mM Tris-HCl (pH 7.6),
10 mM MgCl2, 5 mM DTT.
|
| H2O *** |
|
0-38 mL |
|
| total |
|
50 mL |
|
* Get T4 polynucleotide kinase (Promega) from Core Facilities. It comes with a 10X buffer
** 1 unit is defined as the amount of enzyme required to transfer 1 nmol of phosphate from ATP to the 5'-end of an oligodeoxynucleotide in 30 min at 37 °C with a [ATP]=100 M, [5'-end] = 500 M.
*** Add an appropriate amount of oligodeoxynucleotide solution to get the desired pmol. Add H2O to bring the total volume to 50 L.
|
|
This procedure will produce some labeled oligo, but the majority of oligo will not be labeled. This is OK for most reactions. If you need all the oligos to be 5'-labeled with phosphate then add 2.5 L of
10 mM ATP (25 nmol) and incubate for 5 min. Not longer. If the reaction is incubated longer then a trans-phosphorylase reaction occurs |
- Heat at 60°C for 5 min to inactivate the kinase.
- Remove the unreacted ATP with a spin column.
-
Prepare spin column
- Add 100 mL total volume to the spin column. If volume of
reaction mixture is less than 100 mL, first add reaction to
the top of the column and after it settles into the column
add buffer to total 100 mL.
- Spin for 10 min. Collect eluant in a screw top
microcentrifuge tube.
- with Geiger counter, measure radioactivity in the eluant and
remaining on the column. A good yield will have a 2:1 ratio
of eluant : column.
- Anneal
Add 50% excess of complementary strand.
Heat at 90 C for 2 min and then allow to cool slowly to room
temperature.
|
