
Transfection Conditions: Cell confluency, reagent volume, and post-transfection incubation time are a few key parameters that affect the outcome of transfection experiments. For more information on getting the most out of your transfections, visit our Tips from the Bench section on Optimizing DNA Transfections.
Plasmid DNA and RNA Oligonucleotide Transfection Protocol: The following procedure describes how to perform plasmid DNA and RNA oligonucleotide co-transfections using TransIT-X2® Dynamic Delivery System in 6-well plates. If using vessels with different surface areas, scale accordingly. For more details on performing transfections with TransIT-X2®, please view the full protocol.
A. Plate cells
- Approximately 18-24 hours before transfection, plate cells in 2.5 ml complete growth medium per well in a 6-well plate.
Ideally cells should be ≥80% confluent prior to transfection.
For adherent cells: Plate cells at a density of 0.8 - 3.0 × 105 cells/ml.
For suspension cells: Plate cells at a density of 2.5 - 5.0 × 105 cells/ml.
- Incubate cell cultures overnight.
B. Prepare TransIT-X2®:DNA:RNA complexes (Immediately before transfection)
- Warm TransIT-X2® to room temperature and vortex gently before using.
- Place 250 µl of Opti-MEM® I Reduced-Serum Medium in a sterile tube.
- Add 50 nM guide RNA (final concentration). For example, add 2.5 µl of a 50 µM stock. If using 2-part crRNA + tracrRNA, add at a 1:1 molar ratio. For example, add 2.5 µl of each 50 µM stock. incubate at room temperature for 10 minutes to allow the crRNA and tracrRNA to anneal.
- Add 2.5 µg plasmid DNA encoding Cas9.
- Pipet gently to mix completely.
- Add 5 µl TransIT-X2® to the diluted DNA mixture.
- Pipet gently to mix completely.
- Incubate at room temperature for 15-30 minutes to allow sufficient time for complexes to form.
C. Distribute the complexes to cells in complete growth medium
- Add the TransIT-X2®:DNA:RNA complexes (prepared in Step B) drop-wise to different areas of the wells.
- Gently rock the culture vessel back-and-forth and from side-to-side to distribute the TransIT-X2® Reagent:DNA complexes.
- Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.