Genetique des Interactions Macromoleculaires, Institut Pasteur

Phosphorylation of the oligonucleotide:

Incubate for 30 min. at 37°C.

Inactivate at 85°C for 5 min.

Purification on Spin column G25

The volume of the eluat is about 15 µl to 20 µl.

Primer extension

Use 5 µg of total RNA per assay

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

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% polyacrylamide gel. (the same as sequencing gel). Denaturate at 85°C for 3 min. All the reactions can be performed in a thermo cycler.