Methods.docx - Materials PBS-Eppendorf...

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Materials: PBS - Eppendorf tubes - Hemocytometer - Inverted microscope - Marker pen - 24 well plate - Sterile Micropipette and tips - Pipet aid - Sterile Pasteur pipets - Falcons - Complete medium with serum: e.g., supplemented media with 10% fetal bovine serum (FBS). - Trypan blue (0.4%) - Mammalian cell line - 70% ethanol - Lipofectamine reagentb Methodology: First lab 1- Firstly the old medium was discarded from the cells using micropipettes inside the fumehood. 2- 1 ml of PBS was added for washing the cells. 3- The PBS was discarded using micropipette. 4- 1 ml of trypsin was added to the cells. 5- The cells were incubated at 37c and 5% Co2 for 2 min. 6- Centrifugation was performed at 1200 RPM. 7- 1 ml RPMI media was added to the cells. 8- 100 ul of the cells were transferred into eppindorf. 9- 10 ul of trypan blue were added for counting the cells on 10 ul of cells. 10- 40 ul of the cells were transferred to the 24 well plates. 11- 0.5 ml of complete media was added. 12- The well plate was incubated at 37c and 5% Co2.
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Unformatted text preview: Second lab 1-Two eppindorf were brought and labeled one “DNA” and the other “lipo” inside the fume hood. 2-50 ul of the optimum media were added in both eppindorf using pipette. 3-1.5ul lipofectamin were added in the eppindorf labeled by “lipo”. 4-6.6 ul of DNA were added in the “DNA’ labeled eppindorf along with 2 ul P3000. 5-The content of the “lipo” eppindorf were added to the “DNA” eppindorf using pipette. 6-15 min incubation time passed. 7-The content of the eppindorf was added to the cells in the specific two wells that were in the well plate and they were added drop wise and the plate was gently rotated to distribute the complex. Third Lab 1-The cells were put on the inverted microscope to be seen by the camera on the computer. 2-The cells were seen by 10x lens. 3-The channel was fixed on the blue color to be absorbed and to be able to reflect the green color of the EGFP to be seen....
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