PRIMER LABELLING FOR PRIMER EXTENSION ASSAY

Ulrike Heberlein

PRIMER LABELLING FOR PRIMER EXTENSION ASSAY

Mix in a 20µl final volume: primer labelling;
2µl 10x kinase buffer
1µl oligonucleotide primer; (5pmoles/µl)
200µCi 32P- ATP (crude, ICN, 7000Ci/mmole)
10 U polynucleotide kinase
ddH2O to 20µl
Incubate at 37°C for 30-45 minutes. Heat at 65°C for 15 minutes.
To remove unincorporated label precipitate as follows:
Add 60µl TE + 320µl 2.5M NH4OAc + 20µg RNA carrier + 650µl 100% ethanol (follow this order of addition; don't add the carrier RNA to the reaction first or some RNA will get labelled). Chill on dry ice for 5-10 minutes. Spin in the cold for 15 minutes. Discard supernatant (very hot!!). Resuspend pellet in 180µl TE and add 20µl 3M NaOAc + 500µl 100% ethanol. Chill on dry ice for 15 minutes, spin in the cold for 10 minutes, wash with 70% ethanol, dry and resuspend in 100µl TE (should be ~200cps/µl on minimonitor).

10x kinase buffer:
500mM Tris pH 8
100 mM MgCl2
10mM spermidine
1 mM EDTA
50mM DTT (add fresh before using)