Site-directed mutagenesis, cell culture and transfection

Day one. Set up cells. Cells should be 80-90% confluent. Trypsinize cells for 5 min in a 37°C incubator. Observe the cells under the microscope for detachment. Detached cells will be round and in suspension. Depending on the cell line culture vessel may be gently tapped on the side of the flask. (Note: to avoid clumping do not agitate the cells by tapping while in trypsin.) Then add 10 mL of growth medium and use a 10 mL serological pipette with a narrow mouth on the end to pipette the cell suspension up and down at least 10 times. This step is the most critical to ensure single cells for accurate counting and plating. 12 well dish: 1.5-2X10⁵ cells/well in 1 ml of DMEM/10%FBS

Day two. Transfect cells with siRNAs using Lipofectamine RNAi Max.

1. Prior to transfection bring all reagents to room temperature.
2. In a sterile tube dilute siRNA in Opti-MEM, mix them gently by tapping the bottom of the tube 3 to 6 times.

12 well dish: 50 ml OptiMEM + 10 or 20 nmol of total siRNA  for each well

1. Dilute RNAiMAX in OptiMEM, mix them by gently tapping the bottom of the tube 3 to 6 times. The volume of RNAiMAX used is based on a 3 :1 ratio of RNAiMAX(ml):total siRNA ( 3 ul for 10 mol siRNA, 6 ul for 20 mol siRNA).
2. Combine diluted DNA and diluted RNAiMAX, and gently mix by pipetting up and down 3 to 6 time, gently tapping the bottom of the tube 3 to 6 times, or inverting the tube 3 to 6 times. DO not VORTEX the mixture.
3. Incubate 15 minutes at room temperature.
4. Add siRNA/RNAiMAX mixture to cells drop by drop.
5. Return cells to the CO₂ incubator.

48 to 72 h after transfection, check protein expression by Western Blot.