This lab allows us to transfect DNA into HEK 293 cells, using the CaCl₂ technique.

Materials

Prelab Preparation

Please refer to Lab 1: Making MEM media for Biosafety hood and water bath setup

To start this lab, a cell culture needed to trypsinzed and incubated for 3-5minutes as done many times before.

While the cells are incubating, 6ml of MEM media were transferred to 15ml tube and 4ml of were transferred to all wells of the 6 well plate.

After the incubation period of the trypsin, the 4ml of trypsin and cells were transferred into the 15ml tube with 6ml of media . The cells were then centrifuged and the media was removed using a pasture pipet. The cell pellet was then resuspended in 6ml of MEM media. The cells were lightly vortexed and 10ul were drawn up and deposited in each wedge of the hemocytometer.

With the completion of the calculation above, 155ul of cells were added to all 6 wells of the culture plate. Shown above.

After the 24 hours of incubation, two of the best wells were selected based on confluency. In my case, it was wells 4 and 6 were selected due to having the closest confluency to 40%.

With the wells marked, the media was sucked off and replaced with 2ml of fresh media 2 hours before transfection.

In this lab lab we were provided with 5ul of DNA that could be used to transfect our cells with. With that in mind, two concentrations of DNA solution were made 2ul and 3ul.

Addition of CaCl₂ and 2X HEPES Solution

The entire DNA mixtures were then transferred to their respective wells. The 2ul concentration was spread over well 4 and the 3ul concentration was placed in well 6. The DNA and media was allow to incubate for 11 hours after which the media was removed from all of the wells and 3ml of fresh MEM media were placed in the transfection wells. The incubation was continued for another 24 hours.

On day 3, the media from the transfected wells was removed using a disposable pasture pipette as seen in picture 1. Once the wells were free of media 2ml of 2% of paraformaldehyde were carefully added to both wells . This process needs to be done carefully because the cells could lift off of the plate. after addition the plate was incubated at room temperature for 15 minutes.

While the fixative was incubating , the staining solution was made. This was done by first adding 10ml of PBS followed by 200ul of 250mM potassium ferricyanide, 200ul of 250mM potassium ferrocyanide, 20ul of 2M MgCl₂ and finally 50ul of X-gal 20mg/ml in DMF into a fresh 15ml tube.

Pictures above depict the addition of PBS and Potassium Ferricyanide.

Addition of Potassium Ferrocyanide.

Addition of MgCl₂

Addition of X-gal. X-gal is a substrate that changes the color of cells that were transfected, to blue/green. It allows us to clearly see which cells were transfected and how effective the transfection was.

After the 6 well plate incubated for 15 minutes, the fixative was removed with a disposable pipette. The pipette was disposed of in the biohazard as we were working with a toxic fixative and human cells.

The next step was to add 5ml of staining solution to the wells. Once added the cells were placed into the incubator for 24 hours. After this incubation period, the cells should have a blue cytoplasmic coloration.