West Coast Green Crab Experiment Part 63

RNA library preparation part 2

Sara is walking me through the secoond part of this protocol! Overall it was much easier to run through this part than the first part. Also, I have a very fun magnetic rack that makes it much easier to see the beads!

Notes

  • Picked up some tips from Sara about using a multichannel pipet!
    • Rock the tips onto the pipet when loading
    • When pipetting up small volumes, tilt the tips away from the beads and stick them onto the bottom. Once the tips touch the bottom, keep the tips at the bottom and rock them back and forth to suck up the liquid.
    • When pipetting up large volumes, start sucking up the liquid with the pipet tips angled as they move down into the bottom of the tubes/plate. This prevents the liquid from overflowing.
    • When pipet mixing, only go until the first stop until you’re done mixing and releasing all the liquid
  • Some tips working with beads
    • NEVER LET THE BEADS DRY!
    • Generally, keep the pipet tips angled away from the beads
    • When washing the beads, it helps to pipet the liquid back onto the beads to ensure they are hydrated and mixed back into the liquid.
  • Generally in Sara’s protocol, reagents that don’t fully freeze will not need to be thawed ahead of time, but the should be spun down. For tiny volumes, flick mixing is the best.
  • Made fresh 80% EtOH
  • Kept NEBNext UltraExpress Ligation Master Mix in the freezer until ready to use.
  • Used 11 PCR cycles for PCR enrichment of adaptor ligated DNA
  • Removed 1.3 µL after phased bead cleanup for Qubit dsDNA HS assay.

Results

All primer and cDNA concentration information can be found in this spreadsheet.

One sample had a low yield, but I remember having trouble working with that sample when using a plate for the first day. Sara said she’ll send me a protocol so I can do some additional amplification and improve the yield! Overall, the yields are < 10 ng/µL, so I can increase to 13 PCR cycles at the end.

BUT WE HAVE YIELDS AND THAT IS WHAT MATTERS MOST.

Going forward

  1. Find all RNA samples and move to -20ºC
  2. Move extracted RNA to the -80ºC
  3. Extract RNA
  4. Find all metabolomics samples
  5. Send samples for metabolomics
  6. Try NEB UltraExpress Kit
  7. Order library preparation materials
  8. Examine HOBO data from 2023 experiment
  9. Demographic data analysis for 2023 paper
  10. Start methods and results of 2023 paper
Written on April 22, 2025