a. Fledgling grad student, Joe Peon, is doing PCRand goes to the freezer to get

Question

a.    Fledgling grad student, Joe Peon, is doing PCRand goes to the freezer to get the reagents needed to set up thereaction. This overworked graduate student grabs ddNTPs, 2forward primers, enzyme buffer, and TaqI. out of storage and sets uphis reaction tube. He sets up 2 tubes: 1) the positivecontrol with target DNA added 2) the test sample + targetDNA. He adds all the reagents listed, in the amounts given inthe protocol and sets up the PCR cycler to cycle a heat phase for30 seconds and extend for 1 minute. The next morning, he ranthe samples out in an agarose gel.

                                              i.     What, if anything will Joe Peon see onthe gel?

                                             ii.     What mistakes were made (there arenumerous errors)?

Explanation / Answer

The mistakes are in the primers. There has to be a reverse andforward primer to seal the amplification both at the 5' and3'. The PCR undergoes cycles where there is an extreme heatand cooling period. The the positive control is known sample ofDNA. The second tube should have the master mix plus test sample(DNA he is trying to amplify and compare to the known). He willmore than likely see the positive control, and the combination ofthe second tube. Hope this helps

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