Guidelines for using ProtoScript II RT-PCR Kit

Introduction

First strand cDNA synthesis:
Proper precautions should be used to avoid ribonuclease contamination. This includes the use of autoclaved tubes, baked glassware, ultra-pure solutions, sterile pipette tips and latex gloves during manipulations.

Thaw system components and place on ice. The 10X RT Buffer can be warmed briefly at 45°C and vortexed to dissolve any precipitate. (Note: It is important to set up a –RT control reaction ( no reverse transcriptase) to insure there is not DNA contamination).

Protocol

1. Make the RNA/primer/dNTP mix by combining the following components in a sterile RNase-free microfuge tube:
   
   Total RNA                        1-10 μl (1 ng -1 μg)
   
   Primer dT₂₃VN                 2 μl
   
   dNTP mix                         4 μl
   
   Nuclease-free H₂O          variable
                                    ------------------------
   Total volume                     16 μl
2. Heat for 5 minutes at 70°C. Spin briefly and promptly chill on ice.
3. Add the following components to the 16 μl RNA/primer/dNTP solution and mix well by pipetting up and down:
                                                                           -RT control
   10X RT Buffer                     2 μl                           2 μl
   
   RNase inhibitor                   1 μl                            1 μl
   
   M-MuLV Reverse                 1 μl                             —
   Transcriptase
   
   Nuclease-free H₂O                —                             1 μl
                                          ---------------------------------------
   Final volume                      20 μl                            20 μl
4. Incubate the 20 μl cDNA reaction at 42°C for one hour. If random primers are used, an incubation step at 25°C for 15 minutes prior to the 42°C incubation.
5. Inactivate the enzyme at 90°C for 5 minutes.
6. Bring the reaction to 50 μl with water. The cDNA product should be stored at -20°C.