PCR Protocol for RBr DNA Markers

PCR Reactions with PCR Premix

The following is the procedure for starting PCR reactions for the four individuals in your paternity case and one negative control reaction.  The negative control is a reaction with every ingredient except primers.  This procedure uses a liquid premix that contains Taq DNA polymerase, buffer, magnesium chloride, and the nucleotides dATP, dCTP, dGTP, and dTTP.

We have tried several brands of Taq Polymerase and find that Taq from Syzygy Biotech (www.syzygybiotech.com) give excellent results and is very cost effective.

Part I.  deciding how many microliters of DNA sample to use

In each PCR reaction, you should use 40 nanograms of DNA.  For each sample, calculate how many microliters (rounded to the nearest microliter) that you need to obtain 40 ng.

Consult the instructor if the amount needed is more than 4 µl or less than 1µl.

Part II-A.  setting up a single reaction

1.      Use a fine point Sharpie to label a 200 µl PCR tube with the ID # of the plant.

2.      To this tube add the following.:

            12.5  µl Syzygy Taq 2X Master Mix

              2.5  µl Forward Primer stock

              2.5  µl Reverse Primer stock

                x   µl DNA

           7.5–x µl milliQ water*

                  *The  7.5-x ul of water is added to make the total volume of all reactions equal to 25 µl.

3.      Set a micropipettor on 20 ul and with a new clean tip, gently pipette back and forth twice to mix the reaction components.

4.      Follow your instructor’s direction to load them in the thermal cycler.

Part II-B.  negative control

For each group, prepare one negative control as follows:

            12.5  µl Syzygy Taq 2X Master Mix

              8.5  µl milliQ water

              1.0  µl Mother DNA

              1.0  µl Child  DNA

              1.0  µl Possible Father #1 DNA

              1.0  µl Possible Father #2 DNA

Close the cap on the tube and flick with your finger until the bead is dissolved.  Centrifuge a few seconds to send all of the liquid to the bottom of the tube.  Follow your instructor’s direction to load them in the thermal cycler.

PCR Cycles

94 degrees, 2 minutes

25 cycles of

      94 degrees, 30 seconds

      61 degrees, 1 minute

      72 degrees, 1 minute

72 degrees, 4 minutes

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