Molecular Biology Protocols

 
Marine DNA Sequencing and Analysis Center
Mount Desert Island Biological Laboratory


Image courtesy of Invitrogen

Last update January 13, 2006. 
Send comments to dtowle@mdibl.org

 

5.  Reverse Transcription: SuperScript III First-Strand Synthesis of cDNA

Caution Note:  Cleanliness and care in setting up your experiment are absolutely necessary for useable results.  Calibrate your micropipets daily by weighing water.  Think as you do all of your pipetting.  Do not allow yourself to be distracted during this process.  Observe every pipetting process carefully to make sure you have obtained and dispensed the desired amount.  Wear fresh gloves as you handle the microcentrifuge tubes and set up the reactions.  Think clean!  Use fresh pipet tips for each addition to avoid contaminating your RNA, primers, and reagents.

Thaw, mix and briefly centrifuge each component of the kit before use.  Keep the SuperScript III reverse transcriptase and RNAse on ice.  Refreeze kit components as quickly as possible in a non-frost-free freezer at –20oC.

 

1.        In a nuclease-free 0.2-ml tube, add a volume of total RNA equal to 5 or 2 micrograms.  (5 micrograms is maximum for this system, 2 micrograms is a useful amount for QPCR.)  Keep the RNA on ice as you do this pipetting, and refreeze it in a non-frost-free freezer as quickly as possible.  Treat the RNA like it’s liquid gold!

2.       Add 1 μl  10 mM dNTP mix.

3.       Add 1 μl  Oligo-dT primer.

4.       Add DEPC-treated water to give a total volume of 10 μl.

5.       Incubate at 65oC for 5 min, then on ice for at least 1 min (SS3-1 program in the MJ Research Thermal Cycler).

6.       Prepare reaction mixture as follows:

 

Each reaction               For 5 reactions

            10X RT buffer                          2 μl                              10 μl

            25 mM MgCl2                            4 μl                              20 μl

            0.1 M DTT                                2 μl                              10 μl

            RNaseOUT RNase inhibitor       1 μl                                 5 μl

            Superscript III enzyme            1 μl                                 5 μl

           

            Mix and briefly centrifuge.

 

7.       When step 6 is complete, add 10 μl of the reaction mixture to each RNA/primer mixture.  Mix and briefly centrifuge.

8.       Incubate at 50oC for 50 min (SS3-2 program).  Terminate the reaction at 85oC for 5 min and chill at 4oC (included in the SS3-2 program).

9.       Centrifuge briefly.  Add 1 μl RNAse H to each tube, mix, and incubate for 20 min at 37oC then chill at 4oC (SS3-3 program). 

10.     Store cDNA at -20oC.