|
End-Labelling of Oligonucleotide Primers using
T4 Polynucleotide Kinase
1. Prepare the following mixture in an eppendorf tube:
|
Reagent
|
Amount (µl)
|
|
Primer (24 pmol/µl)
|
1
|
|
32P -ATP (1.85 MBq)
|
5
|
|
10x PNK Buffer
|
3
|
|
T4 polynucleotide kinase (10 U/µl)
|
2
|
|
DEPC water
|
19
|
|
Total
|
30
|
2. Spin briefly in a microfuge and incubate as
indicated below:
|
Temperature (°C)
|
Time (min)
|
|
37
|
30-60
|
|
90
|
5
|
3. Use 1.2 pmol of primer (1.5 µl) per sample (i.e.,
set of four reactions) in the cycle sequencing protocol. The above
is enough for approximately 20 samples.
|
