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FreeStyle™ MAX 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.
1. Count cells (after cell straining if needed).
2. Determine volume needed for 3 × 10
7
cells (per
transfection). Dilute cells with pre-warmed
complete medium to 1 × 10
6
cells/mL in 30 mL;
maintain with shaking at 37°C until needed.
Note: Do not centrifuge cells prior to transfection.
3. Dilute 50 μg of plasmid DNA in 1.5 mL final
volume of OptiPRO™ SFM.
4. Dilute 50 μL FreeStyle™ MAX in 1.45 mL of
OptiPRO™ SFM for each transfection; mix without
vortexing.
5. With tip submerged, slowly add 1.5 mL diluted
FreeStyle™ MAX to diluted DNA; incubate
10–20 minutes at room temperature to allow for
DNA-FreeStyle™ MAX complex formation.
Note: If precipitation is observed, start over from
step 3.
6. Add all 3 mL of FreeStyle™ MAX-DNA mixture
dropwise to cells from step 2 while swirling.
7. Incubate with shaking at 37°C in a humidified
incubator with 8% CO
2
for 2 days and proceed to
Selecting Stable Transfectants for Protein
Expression.
1. a. Cell count (cells/mL):_________________
b. Viability (%):__________________________
2. a. mL cells (3 × 10
7
÷ 1a):_________________
b. mL fresh medium (30 – 2a):____________
3. a. Plasmid ID:__________________________
b. Concentration (μg/μL):_________________
c. μL DNA for 50 μg (50 ÷ 3b):____________
d. μL OptiPRO™ SFM (1500 – 3c):_________
5. DNA + FreeStyle™ MAX complex formation:
Incubation start time:__________________
Incubation end time:__________________
FAQ Sections
Figure 6. FreeStyle
™
MAX transfection protocol and calculations worksheet.
Life Technologies
™
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®
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®
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