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/

Department of Microbiology
School of Medicine
University of Washington
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