10/10/23

10/10/23 kdorfman Mon, 10/02/2023 - 19:38

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