1. Colony Assay: Cells are plated in low number on 6well dishes and allowed to grow over a period of 2weeks. The wells are later stained and compared in terms of cell size and staining density.

2. Western Blot: Cells are lysed and protein is collected. Standard curve is constructed by bradford assay, and the concentration of the protein is calculated. A loding samples is then formed by ensuring that there is 100μg ofprotein in each load. A dye is added to the protein samples and loaded into the wells of the SDS page and ran on 160v for 1hour. The gel is later transferred onto a membrane by sandwiching it between filter paper and sponge with a cassette holding them together and subjected to electrophoresis, 325mA for 2hours. When the transfer is completed, the membrane is washed 3x with wasbuffer for 10mins each and then probed with 5%milk TBS-secondary Ab soolution for 1hour. Afterwhich is washed again 3x wash buffer 10mins each, and then membrane is taken to react with solutioin west pico 1 and 2 to cleave the substrate off the secondary Ab. Finally the membrane is taken to a dark room and exposed for a desired period of time, and processed with a processor, Kodak X-Omat. The xray flim is then analysed, which if bands form, suggest that the particular protein is present and vice versa.

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3. Soft Agar Assay: Cells are embedded in between 2layers of DMEM agar and allowed to grow over 2 weeks. This determines if the respective samples are capable of forming tumors. A positive indication of magliancy can be observed with colonies forming on the agar.

4. Cell Cycle analysis: Cells are just being pass through this FACS analysis machine that can calculate the cell size, cell number, cell granulity, which cell cycle phase the cell is in, etc etc. We are most interested in the cell size and its the stages of the cell cycle that the cell is in.

So far everything has been going pretty well. The xray flims from the western blot of HEK Ras #1 cells showed positive for control and mock while detecting AIP, simply shows that the experiment is least going in the right direction. Very faint bands were formed for AIP which isnt supposed to be the case since the samples were AIP scilenced. this shows that the transfection with siAIP is suscessful, however not enuogh to completely knockdown the AIP gene. Problem might be due to the transfection method, thus more caution should be exercised the next time.

The same membrane was then used to probe for Aurora-A (AIK) protein. With the AIP knocked down, AIK levels should increase, however the bands show equal density as the ones in control and mock. It was then suspected that perhaps the cells are Ras unstable and thus yield lead to the incocnsistancy so then membrane was then probed with Ras-Ab. The results then show that Ras is stable as all 4 samples have the same thick band produced, which indicates Ras stability. We then suspect it might be due to the mechanical issues while performing the experiment, such as incubation time. While the experiment is being repeated, it is necessary to take note of the minute details of the protocol and follow closely to minimize errors.

Even though there were times it got a little tough especially when contamination arises and the cells cannot be used. Hek Ras #3 cells were contaminated after transfection as the cells were incubated for 72hrs before harvesting its proteins. This then wasted 3days of incubation and the western blot could not be derived.

GLAD