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47 Neon ® Transfection Protocol and Calculations Cell culture before transfection: Minimum 5 passages, viability ≥ 95% Note that the numbering below does not match the numbering within the detailed protocol. Prepare Neon ® Transfection System: Set desired voltage, pulse width, and pulse number (record at step 7 below); fill the Neon ® Tube with 3 mL Electrolytic Buffer E2 before inserting it into the Neon ® Pipette Station. 1. Count cells (after cell straining if needed). Determine volume needed for harvesting 4 × 10 7 cells by centrifugation. 2. Centrifuge cells, discard supernatant, and resuspend with at least 20 mL PBS to wash (Wash 1). 3. Centrifuge cells, discard supernatant, and resuspend with at least 20 mL PBS to wash (Wash 2). 4. Recount cells prior to centrifuging Wash 2. 5. Discard supernatant and resuspend cells to 5 x 10 7 cells/mL in Resuspension Buffer R. 6. Mix cells with DNA: generate four tubes containing 120 μL cells + 12 μg DNA OR generate one tube containing 480 μL cells + 48 μg DNA. 7. Without introducing air, draw up 100 μL of cell-DNA mixture into the Neon ® Tip, electroporate, and immediately proceed to step 8. 8. Dispense cells into a T-75 flask containing 10 mL of pre-warmed complete medium (for a total of two T-flasks). 9. Repeat steps 7 and 8 three more times per plasmid, with two repeats dispensed per T-flask, for a total of four repeats and two T-flasks. 10. Incubate T-flasks at 37°C in a humidified incubator with 8% CO 2 for 2 days, pool duplicate T-75 flasks, and proceed to Selecting Stable Transfectants for Protein Expression. 1. a. Cell count (cells/mL):___________________ b. mL cells to centrifuge (4 × 10 7 ÷ 1a):_______ 2. Wash 1 (mL):____________________________ 3. Wash 2 (mL):____________________________ 4. a. Recount (cells/mL):_____________________ b. Total cells (4a × mL Wash 2):_____________ 5. mL Resuspension Buffer R (4b ÷ 5 × 10 7 ):____________________________ 6. Plasmid ID:_____________________________ Concentration (μg/μL):____________________ μL cells (120 or 480 μL):___________________ μL DNA (12 or 48 μg ÷ conc.):_______________ 7. Neon ® electroporation settings: Pulse voltage (V):_________________________ Pulse width (ms):_________________________ Number of pulses:________________________ FAQ Sections Figure 8. Neon ® transfection protocol and calculations worksheet. Life Technologies ™ | Freedom ® CHO-S ® Kit FAQ Download now