Continuing RT and PCR

30/10/09 -

Made an agarose gel consisting of 1.5% (0.75g) agarose with 50ml TBE buffer.

The samples were loaded into the gel with a 1kb ladder. It was run for 1 hour and then the gel was stained with ethidium bromide and was photographed using UV light. Unfortunately the PCR did not work as no DNA was present and only the primers appeared on the gel.

02/11/09 -

RNA was harvested from P.28 cells. This time the TRIzol was put directly into the flask of cells in the hope that this would lyse the cells more efficiently and ensure every cell is collected. This was done in the following way:

- Pour off medium
- Add PBS to wash
- Pour off PBS
**In fume hood**
- Add 1ml TRIzol
- Scrape cells into corner
- Add 500microL to two eppendorfs
- Add 250microL chloroform
-Vortex
Continue extraction process as described previously.

Using the nanodrop the RNA concentration was measured:

RNA concentration - 785.4ng/microL
260/280 - 1.87
260/230 - 0.78

260/280 and 260/230 are both measures of purity. The ideal value for 260/280 should be close to 2 and for 260/230 should be close to 1.

The RNA was then stored at -80 degrees Celsius.

03/11/09 -

cDNA was made from the RNA extracted from the P.28 cells using the same method as previously.

The cDNA was then stored in the freezer.

04/11/09 -

Another PCR reaction was set up in the same manner as before this time with three tubes containing 2 microL cDNA and one tube containing 4 microL cDNA. The final volume was made up to 50 microL using water.

The three tubes were again went through the PCR at three temperatures 50, 55 and 60 degrees Celsius. The tube with 4 microL cDNA was put through the PCR at 55 degrees.

Again I took photos of each flask of cells. Hopefully over time the ageing of the cells will be apparent through these photographs.

05/11/09 -

A 1.5% agarose gel was made and the samples were run for an hour along with a 1kb ladder. The gel was stained with ethidium bromide and photographed under UV light.

Again the PCR did not work, only the primers an primers which had dimerised showed up on the photograph.

This may be because the MRC-5 cells have a low expression of Zmpste24 or that the binding site is right at the end of the 3' UTR and the primers are not binding very well. The next step is to look at the expression of Zmpste24 in these cells as well as a number of other cell lines.

06/11/09 -

Passaged 11 P.28 to 22 P.29 and 2 P.27 to 4 P.28
New medium was added to P.34 and P.35