User:David Benjamin Nyer/Notebook/PcTF breast cancer/2015/11/10

From OpenWetWare

Jump to: navigation, search
PcTF breast cancer Main project page
Previous entry      Next entry

Summary

Neon transfection plan:

BT-474

Sample Well no. Pulse Voltage Pulse width Pulse no.
1A1---
2A2925302
3A3950302
4A4975302
5B11000302
6B21025302
7B31050302
8B41075302
9C11100302
10C21250103
11C31300103
12C41350103

BT-549

Sample Well no. Pulse Voltage Pulse width Pulse no.
1A1---
2A21350201
3A31400201
4A41450201
5B11100202
6B21150202
7B31200202
8B41250202
9C11100301
10C21150301
11C31200301
12C41250301

Methods

  1. Prepare a 12-well plate containing 1 mL culture medium in each well.
  2. Culture cells in T-75 flasks until they reach 75-90% confluency. Wash, trypsinize, and resuspend in fresh medium.
  3. Take a 20 µL aliquot of the resuspended cells and measure cell density using hemacytometer. Calculate the total number of cells in your sample. Transfer 2x105-5x105 cells per sample (3x106-7.5x106 for 12 wells + 3 buffer) into a fresh tube and centrifuge. Resuspend in Resuspension Buffer R at a final concentration of 2x107-5x107 cells/mL (150 µL total).
  4. Add 1 µg of plasmid DNA per sample (15 µg total) to your cells. DNA should be at a concentration of 1-5 µg/µL.
    1. DNA to be used is at 1200 ng/µL, so 12.5 µL of plasmid.
  5. Set up a Neon Tube with 3 mL Electrolytic Buffer into the Neon Pipette Station.
  6. Begin electroporation, using the parameters outlined in the above section.
  7. After electroporation, transfer samples from the 10 µL Neon Tip into the prewarmed 1 mL culture medium.
  8. Rock the plate to ensure even distribution of cells, then incubate at 37°C

Notes

After resuspension in medium, BT-474 density was 1.4E6 cells/mL; BT-549 density was 3.1E5 cells/mL. I spun down 5.35 mL of BT-474 culture and resuspended in 150µL Resuspension Buffer for a final concentration of 5E7 cells/mL. I spun down all 18 mL of BT-549 culture and resuspended in 150 µL Resuspension Buffer for a final concentration of 3.7E7 cells/mL.


Personal tools