DNA Transfection

Purpose

The goal of this lab is to learn the procedure of transfecting DNA into HEK 293 cells.

Materials

HEK 293 cells

MEM media, 10% FBS+PSG

Trypsin/EDTA

15 ml tubes

one 6 well TC Plate

1.5 ml Sterile microfuge tubes

2M CaCl2

Sterile water

2X HBS

5ul of pCMV B-gal at 0.025ug/ul

Pipet aid and pipets

2% paraformaldehyde in PBS

Glycerol

Potassium ferricyanide 250mM

Potassium ferrocyanide 250mM

MgCl2 1 M

X-gal 20 mg/ml in DMF

PBS

Procedure

HEK 293 cells were grew in 10 cm plate near 70% confluency

Media bottle and TE tube were warmed up in water bath at 37 degree Celsius

Biosafety hood and vacuum system were turned up

After rinsing media bottle and TE tube and placing them in hood, media was sucked off cells with pasteur pipet

4ml of Trypsin/EDTA was added to cells and incubated for 5 mins at 37 degree Celsius

While cells were incubating, 6ml of media was added in 15ml tube and 4ml of media in each well of 6 well plate

4ml of cells+TE was transferred to 15ml tube containing 6ml of media

Cells were centrifuged and resuspended in 6ml of media

1.5x10^6 cells were counted to transfer to each well with hemacytometer

Calculation

cells from all four sections: 135 + 150 + 115 + 196 = 596

divide by 4 : 596/4 = 149

Multiply by 10^4: 149 x 10000 = 1,490,000 cells/ml

cells in 6ml in 15ml tube: 1,490,000 cells/ml x 6ml = 8,940,000 cells

To add 1.5 x 10^6 cells: 1.5 x 10^6 cells / 1,490,000 cells/ml = 1.007 ml of cells added

Added 1.5 x 10^6 cells to each well of 6 well plate and grow cells overnight

Next day, two best wells of cells were selected for DNA transfection

0.5-3 hours before transfection, media was removed from cells and fed with 2ml fresh media per well

To prepare DNA, DNA and sterile water were mixed with pipette tip

2M CaCl2 was gently added and mixed

2X HBS was added and mixed by bubble air into mixture 3 times

DNA solution volume per tube/plate:

Solution A:

DNA (2 concentration) = 1ul of 0.25 ug and 2ul of 0.25 ug

CaCl2 = 12.4 ul

Water (sterile) = to make upto 90 ul: 90 - 13.5 = 77.5 and 90 - 14.5 = 76.5

Solution B:

2X HBS = 100 ul

The mixture was incubated at room temperature for 20 min

Then gently bubble mixed and dropped the whole mixture onto cells/media and swirled the plate gently

Incubated the cells/DNA for 2-12 hours

Media was removed and gently washed cells with 3ml of fresh media and continued incubation for 24-48 hours prior to processing for assay

After incubation, media was removed from cells and cells were fixed by adding 2ml of 2% paraformaldehyde in PBS

Incubated at room temperature for 15 minutes

While incubating, prepare staining solution

PBS 10 ml

Potassium ferrocyanide 250 mM 200 ul

Potassium ferricyanide 250 mM 200 ul

MgCl2 1M 20 ul

X-gal 20 mg/ml in DMF 50 ul

Fixative was removed after incubation at room tempearture and then 5 ml of stain solution was added to each well

The plate was incubated at 37 degree Celsius for 24 hours

Result

After 24 hours, pictures of cells/DNA were taken at different magnification and positions and were preserved by covering cells with 2ml of 100% glycerol

Conclusion

In conclusion, this lab experiment trial was successful in transfecting the HEK 293 cells with DNA through DNA transfection.
Cells were stained blue as seen in the pictures above as an evidendence of DNA transfection into HEK 293 cells.
Cells were then preserved using 100% glycerol to maintain a supply of HEK 293 cells.

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