Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 170

25 L. The two strands of the template DNA have to be separated by heating

to 100°C for 5 min in the presence of random oligonucleotide primers.

µ g/ µ

1. Add the following components at room temperature in the order listed:

DEPC-dH 2 O to a fi nal volume of 50 µ L

(taking the entire reaction volume into account) X µ L

Random oligonucleotide primers (hexamers) 1 0.3 A 260 units

Template DNA 25 ng

2. Heat at 100°C for 5 min.

3. Centrifuge for 10 s to recollect sample at bottom of tube.

4. Add at room temperature ( see Note 12 ):

5X Buffer specifi c for DNA polymerase I,

provided with the polymerase by the manufacturer 10 µ L

10 mg/mL Nuclease-free BSA 1 2 µ L

Stock of dATP, dGTP, and dTTP (500 µ M each) 1 2 µ L

[ - 32 P]dCTP (50 µ Ci at 3000Ci/mmol) 1 5 µ L

DNA polymerase I, Klenow fragment 1 5 µ g

Final volume 50 µ L

5. Mix and incubate the reaction tube at the temperature indicated by manufacturer
of the DNA polymerase (room temperature) for 60 min.

6. To terminate the reaction, heat to 100°C for 2 min and chill on wet ice. Add
EDTA to 20 m M .

7. Separate unincorporated oligonucleotides by size-exclusion chromatography
(e.g., Nuctrap Push Columns, Stratagene).

3.5. Hybridization of Probes to Nylon Membranes

1. Wet the dry membrane in TE.

2. Prehybridize for 1-4 h at 42°C in 10 mL of hybridization solution in roller
bottles or containers with tightly fi tting lids. Alternatively, blots and hybridization
solutions can be sealed in plastic bags and put in shaking water bath. Gentle
agitate during prehybridization.

3. Heat radioactive probe for 5 min at 100°C and cool on wet ice. Caution: Use
screw-capped lids to avoid the lid opening under the pressure.

4. Add radioactive probe to hybridization solution at 5 × 10 5 cpm per blot.

5. Hybridize over night at 65°C for riboprobes or at 42°C for cDNA probes with
gentle agitation.

6. Wash twice (30 min each) with 2X SSC, 0.1% SDS at 65°C for riboprobes or at
42°C for cDNA probes with gentle agitation.

7. Wash twice (30 min each) with 0.2X SSC, 0.1% SDS at 65°C for riboprobes or
at 42°C for cDNA probes with gentle agitation.

8. Scan with Geiger counter corners of blot. There should be little to no radioactivity
detectable away from the specifi c bands, if blots are still radioactive. Wash twice
(30 min each) with 0.2X SSC, 0.1% SDS at 65°C for riboprobes or at 42°C
for cDNA probes.