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Bead bashing yeast with Trizol Kit
Cat Triandafillou
24 September 2019
bead-bashing
html_document
pandoc bead-bashing.md -f markdown -t html -o bead-bashing.html -s
pandoc bead-bashing.md -V geometry:margin=0.5in -f markdown -t latex -o bead-bashing.pdf -s

Reagents

Kits

  • Direct-zol RNA Miniprep kit (Zymo cat. no R2072)

Wet reagents

  • Ambion TRI-reagent (Fisher cat. no. AM9738 or similar)

Consumables

  • 1.5 mL eppendorf tubes
  • Zirconia/Silica 0.5 mm Homogenization beads (Biospec cat. no. 11079105Z)
  • Filter tips

Instruments:

  • Microfuge

Notes:

  • Samples are assumed to be flash-frozen pellets of yeast cells from ~1 mL of culture OD 0.1-0.4. For liquid extractions or greater cell numbers, see Direct-zol kit manual for the volume of TRI reagent to add in step 1. The remainder of the protocol will be similar.
  • It is also assumed that samples were frozen in screw-cap tubes -- if not they can be transferred to one after addition of the TRI reagent (step 1).
  • Steps 1-2 should be performed in the fume hood (because of the phenol in the TRI reagent), excepting spins. Once the second collection tube is exchanged for the first in step 2, the rest of the protocol can be done on the benchtop.

Procedure:

  1. Thaw and bash pellets.

    • Add 300 uL of room temperature TRI reagent to each pellet. If cells are not already in a screw-cap or Safe-Lok tube, move them to one now.
    • Add ~1/2 the volume of beads by gentle tapping to dispense from the squeeze bottle they come in -- the exact amount is not critical, but aim to have enough beads that a distinct layer is formed but with enough liquid on top that it may be pipetted off.
    • Put cells in the vortexor on max speed for 10 minutes at room temperature.
    • Briefly spin samples (~30 seconds) at 3000 g.

  2. Precipitate RNA

    • Using a 200uL pipet set to the max volume, transfer liquid from each tube into a new 1.5 mL tube (one per sample, keep samples separate). Try to get as much liquid as possible, avoiding the beads. One or two is okay, but the fewer the better. Try experimenting with different tips and volumes to maximize extraction of the liquid.
    • Add an equal volume (300 uL or whatever volume of TRI reagent you started with) of 100% ethanol and mix thoroughly by pipeting.
    • Transfer all liquid to the columns provided with the Direct-zol kit and spin at ~12 000g for 1 minute.
    • Exchange collection tube (now filled with TRI reagent) for a new one. Dispose with phenol waste (in hood).

  3. Finish following protocol according to Direct-zol manual.

    • Include on-column DNAase treatment if desired.