Primer extension

PRIMER EXTENSION

Kinase of the oligo
1 µl oligo (10 pmoles usualy about 50 ng)
1 µl 10X kinase buffer
1 µl Kinase (10 units Biolabs)
3 µl ATP 32P
4 µl H2O

Incubate for 30 min. at 37°C.
Inactivate at 85°C for 5 min.

Purification on Spin column G25
Put the Spin column G25 in an ependorff tube
Centrifuge the column 1 min. at 3700 rpm in an ependorf centrifuge.
Use another ependorff tube.
Load the 10 µl of kinase reaction on the column (in the middle of the surface of the column)
Centrifuge 2 min at 3700 rpm.
The volume of the eluat is about 15 µl to 20 µl.

Extension primer
Use 5 µg of total RNA per assay
annealing of the oligo and RNA:
O.2 µl radiolabelled oligo (when you use fresh labelled oligo)
1 µl 5XRT buffer
5 µg RNA
Complete at 5 µl with H2O.

Denature at 85°C for 5 min.
Incubate 10 min at 65°C and then 10 min at 42°C
Add 5 µl of the RT mix to each tube
Mix per react:
2 µl 5X buffer
1 µl 100 mM DTT
0.2 µl 25 mM dNTP
0.5 µl Actinomycin D (0.5 µg) (facultatif)
0.5 µl RT. (Invitrogen SuperscriptII)
H20 qsp 5 µl
Incubate the tubes at 42°C for 30 min.
Stop the reaction by adding 6 µl of deionized formamid+Blue (0,05%XC,     0,05%BBP)
Load 8 µl of the reaction on denaturating 5-6% acrylamid gel. (the same as sequencing gel). Denaturate at 85°C for 3 min.
All the reactions can be performed in a thermo cycler