Hot Asymmetric 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 ÂµM) 1.0 Âµl
Template (100ng) 3.0 Âµl
10x PCR Buffer 5.0 Âµl
MgCl2 (25mM) 3.0 Âµl
10X dNTPï¿½s* 5.0 Âµl
32 P dCTP (~10 ÂµCi/Âµl) 10.0 Âµl
Water 22.8 Âµl
Taq polymerase 0.2 Âµl
Final Volume 50.0 Âµl
*10X dNTP stock: 2 mM dGTP/dATP/dTTP and 12 ÂµM dCTP. Final concentration in the PCR reaction is: 0.2 mM dGTP/dATP/dTTP, and 0.0012 mM dCTP. Recipe: combine 5 Âµl 10mM dGTP, 5 Âµl 10mM dATP, 5 Âµl 10mM dTTP and 10 Âµl 30 ÂµM dCTP for 25 Âµl total volume.
5 min at 95Â°C 1X
45 sec at 95Â°C
45 sec at 50Â°C 40X
4 min at 72Â°C
10 min at 72Â°C 1X
hold at 4Â°C
Purify the hot PCR product over a G50 column.
Count 1 Âµl: PCR counts should range ~2-3 x 106 cpm/Âµl
(PRE) HYBRIDIZING THE PCR PROBE
Place blot in hybridization tube: add 6-10ml prehyb/hyb solution.
Place tube in 42Â°C, preheated, hybridization oven. Turn rotator to the ï¿½8ï¿½ setting. Always include a balance tube.
Incubate for 3-6 hours.
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/Âµl of probe (best to use entire probe for 6ml hyb, i.e. 2 x 107 cpm/Âµl).
Pour prehybridization solution out of the hybridization tube and add the hybridization solution containing the probe.
Incubate in the hybridization oven at 42Â°C, rotating, overnight.
Rinse blot 1X quickly at room temp in 2xSSC/0.05%SDS.
Wash 1X for 20 minutes at room temp in 2xSSC/0.05% SDS.
Wash 1X for 30 minutes at 50Â°C in 0.1xSSC/0.1% SDS.
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/