Lab Entry 11 – ISSRs
Two interspersed-simple-sequence-repeat (ISSR) primers were used in PCR reactions.
First, dilutions were made of the template DNA of the plant samples to aid with the success rate when performing PCR. In 1.5 mL tubes, 10 µL of each DNA samples was added to 90 µL of ddH2O to create a 1 : 10 dilution. Two 0.2 mL 8-strip tubes were used where each sample would be placed into one of the tubes on each strip because two PCR tests were run. With all of the tubes labeled with the correct plant ID identification, 1 µL of template DNA was added to each corresponding tube so that both tubes had the exact same elements within them.
Two master mixes were needed in order to run two PCR tests. To make the master mix, we calculated each ingredient to make enough for 20 reactions for my table group so we would have some excess master mix. For both mixes, 250 µL ddH2O, 60 µL 10x bufer + Mg, 20 µL BSA, 40 µL dNTPs, 5 µL Taq, and 5 µL of primer was used to creates the mixes. The primers being used were Omar and 17898 as they displayed the best results after running the samples through gel electrophoresis. Therefore, 5 µL of Omar was added to one master mix and 5 µL of 17898 primer was added to the other master mix. Both mixes were vortexed to ensure the components were mixed well within the tubes.
To each of the tubes PCR tubes containing 1 µL template DNA, 19 µL of master mix was added. Master mix with Omar was placed into a single strip while 17898 was added to the other strip. The tubes were labeled with an “O” or “#” to distinguish between Omar and 17898. The tubes were then placed into separate areas to be run through PCR.
