Testing Zymo cfDNA Kit on LCM Samples

5/28/24 - Extracting DNA from LCM-captured cells (LCM date 5/8/24)

Used MAGicBead™ cfDNA Isolation Kit, protocol

Sectioning performed on 5/6/24 and LCM on 5/9/24. See details here

Protocol notes:

• Followed the protocol almost exactly, but did all incubations at 56 ºC based on other Paxgene extraction protocols. Made the following notes:
  • Resuspended cells in 200 uL DNAse/RNAse free water. Flicked to mix and spun down in mini (benchtop) centrifuge for 2 minutes
  • Transferred contents to 1.5mL tube with 50uL of digestion buffer and 8uL of Proteinase K, following instructions for 200uL input
  • Vortex and spun down, incubated in thermomixer
    • 15 minutes at 55 ºC
    • 15 minutes at 37 ºC
  • Added 50 uL binding buffer and vortex, add 10 uL of resuspended beads and vortex, put on rotator for 5 minutes at 30 rpm
  • Flick tube, open cap, pellet on magnet for 2 minutes
  • Discard supernatant and remove tube from stand
  • Add 800 uL of wash buffer and transfer to clean 2 mL tube
    • did another wash of 800 uL instead of 300 uL
    • then a final 300 uL wash
  • Did two elutions of 15 uL into separate tubes, quantified seperately
• Used the following two samples
  • LCM sample #1
  • LCM sample #8

Qubit Results

• Used High Sensitivity DNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 62.32 (S1) & 22223.15 (S2)

colony_id	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
LCM sample #1, Elution 1	0.125	0.122	0.124 ng/uL
LCM sample #1, Elution 2	nd	nd	0 ng/uL
LCM sample #8, Elution 1	0.865	0.850	0.858 ng/uL
LCM sample #8, Elution 2	0.172	0.174	0.173 ng/uL

DNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

Loaded 10 uL of extracted DNA (Elution 1 only) into each well (3&5 ) – no DNA present.