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

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Summary

Neon transfection plan:

BT-474

Sample Well no. Pulse Voltage Pulse width Pulse no.
1 A1 - - -
2 A2 925 30 2
3 A3 950 30 2
4 A4 975 30 2
5 B1 1000 30 2
6 B2 1025 30 2
7 B3 1050 30 2
8 B4 1075 30 2
9 C1 1100 30 2
10 C2 1250 10 3
11 C3 1300 10 3
12 C4 1350 10 3

BT-549

Sample Well no. Pulse Voltage Pulse width Pulse no.
1 A1 - - -
2 A2 1350 20 1
3 A3 1400 20 1
4 A4 1450 20 1
5 B1 1100 20 2
6 B2 1150 20 2
7 B3 1200 20 2
8 B4 1250 20 2
9 C1 1100 30 1
10 C2 1150 30 1
11 C3 1200 30 1
12 C4 1250 30 1

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.