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Barcoding the DNA (Week 9)

Last week I continued to try to figure out why the barcoded samples weren't working. I tried different annealing temperatures, different amounts of DNA, different DNA samples and different reagents to help it work... And the DNA still wouldn't amplify with the barcoded primers. Finally, I did a PCR where I diluted the barcoded primers. After so many disappointments, I wasn't expecting it to work, but to my surprise, it did.

After figuring that out, I spent the rest of the week amplifying and barcoding all my DNA samples, as well as samples from other people in the lab. In the end, we had enough samples to fill a 96-well plate with only a few empty spaces. Unfortunately, none of the cave samples amplified with the Archaea or Fungal barcoded primers. However, this wasn't totally unexpected, because there doesn't seem to be much of either of those in the cave samples.

For the deep sequencing, each of the DNA samples had to be barcoded with a unique barcode. This meant that each DNA sample needed to have a unique set of forward and reverse primers when it was amplified. So, as I was filling the PCR tubes to amplify my samples, I had to be very, very careful to put one microliter (a very small amount!) of exactly the right two primers into each of the many, tiny tubes that were lined up in the hood. Combined with the fact that I had to stop the PCR machine after 20 cycles each time in order to add the barcoded primers, it became the most stressful experiment I have performed yet! All the samples are amplified and barcoded now.