7 In Situ Hybridization for RNA: Radioactive DNA Probe
103
Procedure
Labeling probe (Nick translation)
There are many kinds of probes such as eDNA, RNA and oligonucleotides and also many kinds oflabeling methods, nick translation , end labeling and random labeling. Researchers should
select the most suitable radioisotope, probe and labeling method, to obtain an excellent result for each study. We will introduce
one labeling method called « nick translation « which we have
used.
1. Put 100 /lCi (in 100 /ll 50% ethanol) of3H-dTTP (or CTP) Nick
into an Eppendorf tube
translation
2. Remove ethanol by speed vacuum concentrator (Make several holes with a pin on the cover of the tube)
3. Add the remaining reagents (on ice) to the tube in the following order:
H2O
Mix by Vortex for 2 mins
DNA Illg
5 x NT buffer
dTTP
Mix by Vortex briefly
DNase I (1:750 (mg/ml))
DNA polymerase I (15 units)
Spin for 1 sec
Total
5 x NT (nick translation) buffer
- 0.25 M Tris-HC1, pH 7.9
- 25 mM MgCIz
- 50 mM E-mercaptoethanol
X III
Y III
10 III
5 III
4 III
3 III
50 III
- BSA (Miles, Pentax) 250 /lg/ml. Store at -20°C