Griffin:Nested RT-PCR

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What is nested PCR?

Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair generates an amplicon within the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first amplicon and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.

Primer Tm Values

Tm values for PCR primers generally 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.

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.

cDNA Synthesis (Reverse Transcription)

  • 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

  • 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 μ 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)

First PCR reaction

  • 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

  • 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.

Second PCR reaction

  • 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

  • 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 by agarose gel electrophoresis stained with an appropriate dye.
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