Guidelines for PCR Optimization Using ProtoScript II RT-PCR Kit

Introduction

We recommend 2-5 μl of the diluted cDNA product per 50 μl PCR reaction.

Protocol

1. Mix the following in a PCR tube on ice:
   Taq 2X Master Mix                 25 μl (mix well by inverting before use)
   
   Forward Primer (10 μm)         1 μl (final concentration 200 nm)
   
   Reverse Primer (10 μm)         1 μl (final concentration 200 nm)
   
   Diluted cDNA                         2-5 μl
   
   H₂O                                      variable
                                             ___________
   Total volume                          50 μl
2. Mix gently. Overlay with mineral oil if the thermal cycler lacks a heated lid.
3. The following PCR cycling conditions are recommended for 0.2 ml thin-wall PCR tubes on BIO-Rad icycler or similar thermocyclers. For other PCR tubes or cyclers, it may be necessary to modify the cycle times:
   
   Initial Denaturation       95°C         1-2 minutes
   
   25-35 cycles                94°C         30 seconds
                                    45-68°C     10-30 seconds
                                     68°C         1 minute per kb
   
   Final Extension             68°C         5-10 minutes
4. Analyze 5 μl of the PCR product by agarose gel electrophoresis.
5. The following control reactions can be used to examine the quality of kit components and RT-PCR products by the kits. The positive control reaction should give a 327 bp fragment, and no product is detectable in the –RT Reaction (figure 3). If the PCR product is deteced in the –RT control reaction, it is due to either the contamination of genomic DNA or a carry-over PCR product (see troubleshooting guide).
   
                                      Positive Control                  -RT Control
   10X RT Buffer                 2 μl                                         2 μl
   
   RNase Inhibitor               1 μl                                         1 μl
   
   Rat Liver Total RNA         1 μl                                         1 μl
   (500 ng/μl)
   
   dT₂₃VN (500 μm)            2 μl                                         2 μl
   
   dNTP mix (2.5 mM)         4 μl                                         4 μl
   
   M-MuLV Reverse            1 μl                                           —
   Transcriptase
   
   Nuclease-free H₂O          9 μl                                         10 μl
                                        _____________________________
   
   Total volume                 20 μl                                        20 μl
6. Mix well by pipetting and incubate at 42°C for one hour.
7. Inactivate the reverse transcriptase by heating at 90°C for 5 minutes.
8. Dilute the cDNA by adding 30 μl H₂0 and add the diluted DNA to the following PCR reaction:
   Taq 2X Master Mix                            25 μl
   
   GAPDH Primer Set (10 μm)                 1 μl
   
   Diluted cDNA                                     2 μl
   
   H^₂O                                                 22 μl
                                                    _____________
   Total volume                                   50 μl
   
   The following PCR cycling conditions are recommended:
   
   Initial Denaturation         94°C         2 minutes
   
                                       94°C         30 seconds
   30 Cycles                      55°C         15 seconds
                                       68°C         30 seconds
   
   Final Extension               68°C         5 minutes
9. Analyze 5 μl of the reaction on a 1% agarose gel, stained with ethidium bromide.