Labeling (Random Priming) of the Probe - French method
Thaw the samples and work always on ice and behind glass.
1. Add template DNA (50-100ng) to ddH2O (final volume 42l ) and boil 7 minutes to denature.
2. Meanwhile, pipet together on ice, in the following order:
10l OLB (Oligo Labelling Buffer)
2l BSA (10mg/ml)
1l Klenow (polymerase I)
5l a 32P (dCTP)
3. Add boiled DNA/H20 to above mixture and incubate at 37° C for one hour (minimum).
4. Purify the labeled probe by ProbeQuant G-50 Column Method (below).
5. Immediately prior to use, boil purified probe for 7 minutes to denature. Cool briefly on ice; pipet into prehybridization solution in hybridization bottle.
Purification of the Labelled Probe
OLB (Oligo Labelling Buffer)
Solution 0 = 1.25 M Tris-HCl, 0.125 M Mg Cl2, pH 8.0 - store at 4°C
Solution A =
solution 0 1 ml
ß-mercaptoethanol 18 Ál
dATP 5 Ál
dTTP 5 Ál
dGTP 5 Ál
Each triphosphate is previously dissolved in TE (3 mM Tris-Cl, 0.2 mM EDTA)
Store at -20°C
Solution B = 2 M Hepes, titrated to pH 6.6 with 4 M NaOH - store at 4°C
Solution C = Hexadeoxyribonucleotides pd(N)6 sodium salt from PharmaciaBiotech (50 units). Resuspend in TE at 90 OD U/ml # 560 Ál (=555 Ál) = (50 U)
Store at -20°C by aliquots
Mix solutions A, B and C in a ratio 100 (373 Ál), 250 (933 Ál), 150 (560 Ál)
To an eppendorf tube add the following:-
DNA (25 ng) - 1 ul
d H2O - 8 ul
Heat for 10 min. at 95oC and chill immediately on ice.
Then, add the following:-
d CTP (0.5 mM) - 1 ul
d GTP (0.5 mM) - 1 ul
d TTP (0.5 mM) - 1 ul
Reaction mixture - 2 ul
a 32P dATP - 5 ul
Klenow (5 u/ul) - 1 ul
Incubate at 37oC for 30 min.
Run on a G-50 column to separate free radioactivity from the labeled probe.
NOTE:- If not using the kit, make up 10x Klenow buffer and use it instead of the reaction mixture.
1x Klenow buffer - 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 7.5 mM DTT