HOT ASSYMETRIC PCR (KATZE LAB)

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:

4.  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).

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

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

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

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

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

10.  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/

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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