RNA isolation

Today I began the process of isolating RNA from the “official” crab hemolymph samples, although they can change if I mess things up, because there are many extra samples.

There are going to be 56 total samples (which will be pooled into 14), so I separated them into 7 groups of 8. img

Mistakes were made during the first batch

I processed the first batch of 8, however at the last step of centrifuging in the protocol (Step 17) I realized I was off by a zero. Meaning, that when the protocol called for centrifuging at 12,000g, I actaully did it at 1,200 g. I’m not sure if this was the case the entire protocol through, or if I accidentally bumped it at some point.

Regardless, I asked Sam what this means in terms of the potential success of the RNA isolation and he said that it could potentially be bad. The RNA needs to be spun at a certain speed in order to come out of solution, so if I was spinning them too slow, it could have been unsuccessful.

I ran Qubit on samples 56 (infected, first sampling day) and 176 (uninfected, first sampling day). The sample amount that I used was 10µL (last time i did Qubit on the test samples, I did 5µL of sample).

The results are as follows:

  • tube 56: 0.896 ng/µL
  • tube 176: Out of range img

I can pick new samples and re-do it. I created a GitHub issue to see what the others think.

Written on February 28, 2018