| NOTE: Tm values for PCR primers offered by Santa Cruz Biotechnology Inc. range between 55-60 C (19-21 nt, GC% ~55%). The A and B nested primer sets share similar base pair length, GC% and Tm values. |
| NOTE: Nested PCR utilizes two pairs of PCR primers for a single locus. The first primer pair A set amplifies within the locus. The second primer pair B set (nested primers) then binds within the "A" amplicon to produce a second nested "B" amplicon. |
| • |
Prepare a solution containing -
| a) 1 μl oligo (dT)12-18 (500 μg/ml) |
| b) 1 ng-5 μg total RNA |
| c) 1 μl 10 mM dNTPs |
| d) and add RNase-free water to a final volume of 12 μl |
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| • |
Incubate at 70° C for 5 minutes to minimize RNA secondary structure, quick chill on ice and then add -
| a) 4 μl 5x reverse transcriptase buffer |
| b) 2 μl 0.1 M DTT |
| c) 1 μl RNase inhibitor |
|
| • |
Incubate at 42° C for 2 minutes to anneal primer and template. |
| • |
Add 1 μl reverse transcriptase (200 units) and incubate at 42° C for 50 minutes to extend the primer and then terminate the reaction by incubating at 70° C for 15 minutes. |
| NOTE: (As an optional step add 1 μl RNase H (2 unit/μl) and incubate at 37° C for 20 minutes) |
| • |
Prepare a solution containing -
| a) |
5 μl 10x PCR buffer (with or without* MgCl2) |
| b) |
*5 μl 25 mM MgCl2 (It may be necessary to vary the MgCl2 concentration, 2.5 mM final concentration recommended.) |
| c) |
1 μl 10 mM dNTP |
| d) |
1 μl primer pair A |
| e) |
1 μl Taq DNA polymerase |
| f) |
2 μl cDNA and add water to 50 μl |
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| • |
Incubate at 94° C for 2 minutes to denature the cDNA. |
| • |
Perform 15-40 cycles of PCR. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. |
| • |
Prepare a solution containing -
| a) |
5 μl 10x PCR buffer (with or without* MgCl2) |
| b) |
*5 μl 25 mM MgCl2 (It may be necessary to vary the MgCl2 concentration, 2.5 mM final concentration recommended.) |
| c) |
1 μl 10 mM dNTP |
| d) |
1 μl primer pair B |
| e) |
1 μl Taq DNA polymerase |
| f) |
1-5 μl first PCR product and add water to 50 μl |
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| • |
Incubate at 94° C for 2 minutes to denature the cDNA. |
| • |
Perform 15-40 PCR cycles. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. |
| • |
PCR products are visualized on agarose gels stained with ethidium bromide. |
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Semi-Quantitative Nested RT-PCR |
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