Home Protocols Guidelines for using Phusion RT-PCR Kit

Guidelines for using Phusion RT-PCR Kit

Introduction

1. Guidelines for reverse transcriptase:

  • Use gloves and RNase free plastic ware to prevent RNase contamination.
  • Prepare premixes to avoid pipetting very small volumes.
  • Pipet all components on ice.
  • Reaction volume in the cDNA synthesis is 20 μl.
  • Use up to 1 μg of RNA template. The minimum amount depends on both the template     and the primers used.
  • Recommended primer amounts in a 20 μl reaction:
    • 100 ng oligo(dT) primers (can be increased up to 1 μg or
    • 50 ng random primers (may require optimization) or
    • 5 pmol (2-10 pmol) gene-specific primers.
Note: When determining the amount of RNA template, the expression level of the target RNA molecule should be considered, as it affects the subsequent PCR step. The volume of the cDNA reaction mixture used as a source template in PCR should not exceed 10% of the final PCR reaction volume. A high RNA concentration in the PCR may also inhibit the reaction.

Protocol

  1. Thaw template RNA, 10X RT Buffer, dNTPs and primers. Mix the individual solutions to assure homogeneity and centrifuge briefly before pipetting.

  2. Combine the following components in reaction tubes
        Template RNA             x μl (up to 1 μg)
        10 mM dNTP mix         1 μl
        oligo(dT) primer*       1 μl
        RNase-free H2O         add to 10 μl

    *Alternatively, random primers (1μl of the 50 ng/μl stock, provided with the kit), or gene-specific primers (volume depends on the concentration of the primer stock) can be used.

  3. Incubate at 65°C for 5 minutes to predenature the RNA.

  4. Place the reaction tubes on ice and add to each tube
        10X RT Buffer            2 μl
        RT enzyme mix          2 μl
        Rnase-free H2O          6 μl

  5. Program the thermal cycler as outlined in table 1.

    Table 1.     Cycler protocol for cDNA synthesis.
    Step Temperature Time
    Primer extension 25°C 10 min
    cDNA synthesis 40°C 30 min
    Reaction termination 85°C 5 min
    Cooling of the sample 4°C Hold


  6. Place the tubes in the cycler and start the program.