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
- Prepare a 12-well plate containing 1 mL culture medium in each well.
- Culture cells in T-75 flasks until they reach 75-90% confluency. Wash, trypsinize, and resuspend in fresh medium.
- 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).
- 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.
- DNA to be used is at 1200 ng/µL, so 12.5 µL of plasmid.
- Set up a Neon Tube with 3 mL Electrolytic Buffer into the Neon Pipette Station.
- Begin electroporation, using the parameters outlined in the above section.
- After electroporation, transfer samples from the 10 µL Neon Tip into the prewarmed 1 mL culture medium.
- 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.
|