Radiolabeleing

1. set up reaction as follows:

2 ul of 10 uM oligo

2 ul of T4 polynucleotide kinase 10X ligase buffer

10 ul H2O

1 ul T4 polynucleotide kinase

5 ul g-32P ATP

20 ul total

3. Incubate 45 min. at 37°C

4. Heat inactivate polynucleotide kinase for 10 min. at 70°C

5. Phenol/chloroform extract oligonucleotide*

add 30 ul of water to bring volume up to 50 ul

add 50 ul phenol/chloroform; vortex

take aqueous layer

6. G-25 sephadex purify labeled oligonucleotide

a. resuspend column by inverting; take off top and cap

b. let column buffer elute by gravity

c. tape tube to column, cover top w/ parafilm, place entire assembly in 15 ml tube

d. centrifuge 2 min 1100xg (2200rpm) to dry column

e. load all of the phenol/chloroform extracted oligo, making sure it is loading in the

center

f. centrifuge in 15 ml tube for 2 min at 2200 rpm

g. transfer eluted oligonucleotide* to new microfuge tube

7. dilute 2:100 ul H2O of recovered oligo in new tube

8. spot 2 ul onto filter paper; place in plastic tube and cap

9. read cpm in scintillation counter. use black button for 32P. 1 min read time.

UPR, Adj. Disc

10. calculate radioactivity

11. anneal half of the labeled oligo with 2X the number of unlabeled oligo of the opposite

strand

25 ul of labeled oligo (0.5 uM)

2 ul of 10 uM unlabeled oligo of the opposite strand

place mixture at 90°C, turn off water bath to let it cool overnight