DNA-DNA HYBRIDIZATION OF 32P LABELLED PROBE

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

resuspend the resin in the column by vortexing
loosen the cap 1/4 turn and snap off the bottom closure
place the column in a 1.5ml microcentrifuge tube for support
pre-spin the column for 1 minute at 3,000rpm
place the column in a new 1.5ml tube
slowly apply the mixture to the top of the resin, being careful not to disturb the

resin bed

spin the column at 3,000rpm for 2 minutes
the purified sample has collected in the bottom of the support tube — this is the 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 µl

dATP 5 µl

dTTP 5 µl

dGTP 5 µl

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 µl (=555 µl) = (50 U)

Store at -20°C by aliquots

Mix solutions A, B and C in a ratio 100 (373 µl), 250 (933 µl), 150 (560 µl)

Store OLB at -20°C

RANDOM PRIME-LABELING-PAM

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