Molecular Biology Protocols

 
Marine DNA Sequencing and Analysis Center
Mount Desert Island Biological Laboratory


Image courtesy of MJ Research.

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

 

6.  Polymerase Chain Reaction Amplification of cDNA Templates

Caution Note:  Cleanliness and care in setting up your PCR 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 stock cDNA, primers, and reagents.

  1. The shipping information for each of your primers will contain the amount synthesized in picomoles.  Dissolve your PCR primers in sterile 1X TE buffer to give 100 picomoles/ml for degenerate primers and 25 picomoles/ml for non-degenerate primers.  (For example, if you are provided 22 nanomoles of primer in a vial, add 880 ml of 1X TE to the vial to produce 25 picomoles/ml.)  Make sure the primers are completely dissolved by vortexing. Store on ice until use and in the -20C freezer between uses.
  2. If you are starting with cDNA from the First Strand Synthesis reaction, use 1 ml of that product as template for each 50-ml PCR reaction. You should design your PCR experiment before proceeding!  Plan to use all of your primers in all reasonable pair-wise combinations. Here is a form that may help you plan:

Tube No.

Forward Primer

Reverse Primer

cDNA Template

1

 

 

 

2

 

 

 

3

 

 

 

4

 

 

 

5

 

 

 

6

 

 

 

7

 

 

 

Etc.

 

 

 
  1. In a 1.7-ml microcentrifuge tube, mix a PCR "cocktail" as follows, using the Sigma RedTaq ReadyMix materials:

Component

ml per reaction

multiply ml by number of reactions + one

Sigma RedTaq ReadyMix (contains buffer, nucleotides, MgCl2, and taq DNA polymerase, as well as loading dye for electrophoresis)

25

 

Nuclease-free H2O

23

 
  1. Add 48 ml of the PCR cocktail to each of your PCR reaction tubes (0.2-ml tubes or tube strips).
  2. Add 0.5 ml of one forward primer and 0.5 ml of one reverse primer to each tube, using a fresh pipet tip for each addition.  Dispense primers into the 48 ml of cocktail rather than onto the side of the tube or into the air above the solution.  
  3. In the same way, add 1 ml of cDNA template to each tube, using a fresh pipet tip for each addition.
  4. Tightly cap the tubes.  Mix the contents thoroughly by tapping vigorously with a finger and spin the tubes briefly in a microcentrifuge to collect the material in the bottom of the tubes.
  5. Place the tubes in the thermal cycler and start one of two programs:

DAVID45 for degenerate primers

DAVID55 for non-degenerate primers

Both programs will incubate the samples according to the following protocol: 92C for 1 min, 45 or 55C for 1 min, 72C for 2 min, cycle back to step one 29 more times, then 72C for 5 min to extend any short products, holding at 4C indefinitely (until we place the tubes in the freezer for electrophoresis later). If we fail to obtain reasonable products with these protocols, then we can try different annealing temperatures other than 45 or 55C. Or we can try varying the MgCl2 concentration. Or we can try different primers.  Or....