AllPrep protocol PDX tissue 2021-01-27

Prep

  1. Make up 70% EtOH (1ml/sample)
  2. Add β-ME to RLT+ buffer. Make up 10ml at a time (add 100ul of β-ME to 10ml of RLT+). Make up fresh if >1 Month old

Labels

  1. DNA final 1.5ml collection tube.
  2. RNA final 1.5ml collection tube

DNA

  1. Cut tissue with disposable scalpel while frozen in cryotube. Move tissue to tube suitable for TissueLyzer. Aim for no more than 30mg. Weigh all tubes in batch, adjusting if necessary
  2. Add bead + 600ul of RLT plus.
  3. Disrupt using TissueLyser
  4. Centrifuge in tabletop centrifuge for 3 min at max speed
  5. Move supernatant carefully into AllPrep DNA spin column. Spin for 30 sec at 10k rpm.
  6. Move AllPrep DNA column to a new collection tube and place at 4C (for later purification). SAVE flowthrough for RNA extraction below!!!

RNA

  1. Add 600ul 70% EtOH and mix well by pipetting

  2. Transfer 700ul to RNeasy spin column placed in 2ml collection tube. Centrifuge at 10k rpm for 15sec.

    1. Repeat if necessary
    2. Discard flow-through

  3. Add 700ul Buffer RW1 to RNeasy spin column. Centrifuge 15sec at 10k rpm. Discard flowthrough-

    empty collection tube completely!

  4. Add 500ul Buff RPE. Centrifuge 15sec at 10k rpm. discard flow through

  5. Place in new collection tube, centrifuge at full speed for 1min