10/10/23

Kate transfects HCT116 cells with Eb1 plasmids.

Need 8-10 viewing dishes, seeded with transfected cells 1 to 2 days before viewing.

1 reaction needs ~ 10⁶ cells/mL

100 uL cells per reaction (barely 8 drops)

So do 2 reactions, each with 10⁶ cells.

2 days before, seed a 25 cm² flask with 0.5 10⁶cells.

(4 days before, seed with 0.125 10⁶ cells)

1 drop of the 100 uL reaction into its own dish.

On transfection day, divide into 2 reactions

Notes on actual protocol followed:

Friday 10/6

• Start with 2 confluent 12.5 cm² flasks
  • (~1.25 x 10⁶ cells)
  • Need 1-5 x 10⁶ cells/mL on Monday
  • Split 1/8 to allow for 3 doublings
  • Stop reaction with 1.6 mL
  • Seed 0.2 mL into each of 2 new flasks.

Monday 10/9/23

• Put all the cells from one confluent 12.5 cm² flask
  • should be confluent again
• into a new 25 cm² flask

Tuesday 10/10/23

• There should be 2.5 x 10⁶ cells
• Trypsinize
• Stop reaction with 2 mL
• split suspended cells into two microfuge tubes.
• Spin down, and proceed with 2 Mirus nucleofection reactions
• Make 2 12.5 cm² flasks