Primary PCR should be performed with the Unigene forward and reverse primers. Purify PCR product using Millipore MAFBNOB10 plates. Elute in water, and use the purified product as a template in the next reaction. Note that you use only one primer (VNG26), in order to make the complementary strand alone. This is a linear, asymmetric PCR reaction.

Combine reagents listed below:

Primer VNG26 (30 µM) 1.0 µl

Template (100ng) 3.0 µl

10x PCR Buffer 5.0 µl

MgCl₂ (25mM) 3.0 µl

10X dNTP�s^* 5.0 µl

³² P dCTP (~10 µCi/µl) 10.0 µl

Water 22.8 µl

Taq polymerase 0.2 µl

Final Volume 50.0 µl

*10X dNTP stock: 2 mM dGTP/dATP/dTTP and 12 µM dCTP. Final concentration in the PCR reaction is: 0.2 mM dGTP/dATP/dTTP, and 0.0012 mM dCTP. Recipe: combine 5 µl 10mM dGTP, 5 µl 10mM dATP, 5 µl 10mM dTTP and 10 µl 30 µM dCTP for 25 µl total volume.

PCR cycles:

5 min at 95°C 1X

45 sec at 95°C

45 sec at 50°C 40X

4 min at 72°C

10 min at 72°C 1X

hold at 4°C

Purify the hot PCR product over a G50 column.

Count 1 µl: PCR counts should range ~2-3 x 10⁶ cpm/µl

(PRE) HYBRIDIZING THE PCR PROBE

Prehybridization:

1. Place blot in hybridization tube: add 6-10ml prehyb/hyb solution.

2. Place tube in 42°C, preheated, hybridization oven. Turn rotator to the �8� setting. Always include a balance tube.

3. Incubate for 3-6 hours.

Hybridization:

1. Boil labeled probe for 2 minutes. Chill on ice for 2 minutes, and add to the hybridization solution. Mix well before pouring the solution into the tube containing the blot. Use 6ml of solution with at least 2 x 10⁶ cpm/µl of probe (best to use entire probe for 6ml hyb, i.e. 2 x 10⁷ cpm/µl).

2. Pour prehybridization solution out of the hybridization tube and add the hybridization solution containing the probe.

3. Incubate in the hybridization oven at 42°C, rotating, overnight.

4. Rinse blot 1X quickly at room temp in 2xSSC/0.05%SDS.

5. Wash 1X for 20 minutes at room temp in 2xSSC/0.05% SDS.

6. Wash 1X for 30 minutes at 50°C in 0.1xSSC/0.1% SDS.

7. Wrap in Saran while the blot is still damp, and expose with a phosphor image screen.

Purchasing Bacterial Clones for Northern Verification:

Purchase the clones by their IMAGE ID number. The vendor is Research Genetics (phone number 1-800-533-4363) The cost is $45 for sequence verified clones.

Search engine with more clone info: http://www.resgen.com/resources/apps/cdna/index.php3 (Type in the IMAGE ID).

Image website: http://image.llnl.gov/