Taqman results, Plasmid DNA purification and re-transformation

2/12/2009 -

The Taqman assay showed each of the cell lines expressed Zmpste24 at differing levels. The assay worked well which is encouraging as we were unsure that the RNA was of a good quality.

The results were analysed using Excel. All cycles in 18S should be amplifying within 1.5 cts (cycle thresholds) of each other, any that were not were removed from the analysis. These cell lines were unlikely to have RNA that was of sufficient quality. Cycles of Zmpste24 should not be amplifying at the same time as we want them to be different.

A western blot was run using antibodies specific for GAPDH, a protein found in all cells. This is run as a control to make sure the same amount of protein was run in each lane. The antibodies were annealed to the membrane and proteins in the same way as before. Then 9ml of water and 1 ml of chemiluminescent was poured onto the membrane and left for 1 minute. The membrane was then put into a light-proof case and exposed with an AUTO-RAD sheet for 1 minute then developed. The resulting picture showed that there was the same amount of P.29 and P.30 protein loaded in the wells, in fact maybe a little more of the of the P.30.

1 colony had grown on the agar plate with both the vector and insert. Using a pipette tip the colony was scrapped and transferred into into 5ml of LB with 100 microG of ampicillin. This was then incubated over night at 37 degrees Celsius shaking.

3/12/2009 -

The Plasmid DNA was purified using the QIAprep spin mini-prep kit as per the provided protocol.

The purified DNA was then digested to check the insert had been incorporated into the plasmid, as follows:

2 microL DNA
2 microL H3 enzyme
2 microL H3 buffer
14 microL water

Then incubated at 37 degrees Celsius for 1 hour.

An electrophoresis gel was then ran using 5 microL of a 1kb+ ladder and 10 microL of the digested DNA. This was left to run at 110V for 1 hour.

The gel was then stained with ethidium bromide and a gel picture was taken. The vector and insert could not be seen, this may be due to a low concentration of DNA. A nanodrop was used to measure the DNA concentration:

-15.4 ng/microL

This is a very low yield usually you would expect a yield of around 200ng/microL using the QIAgen prep kit.


4/12/2009 -

Cells were counted using a haemocytometer. After counting it was seen there had been 2.4 population doublings. 300,000 cells were passaged to P.32.

The other flask was used to harvest cells to extract RNA and protein. This was done using the same method as previously.

7/12/2009 -

The clone with the low DNA yield was re-transformed. Three colonies were picked and transferred into LB + ampicillin and stored in the shaking incubator over night at 37 degrees Celsius.

8/12/2009 -

The DNA concentration of each of the three colonies was tested using the Nanodrop: -
1) 106.6 ng/microL
2) 123.9 ng/microL
3) 140 ng/microL

These concentrations meant there is a better yield of DNA and so a digestion would be more efficient.

Digestion of the plasmid DNA was done as follows: -

4 microL DNA
2 microL H3 buffer
2 microL H3 enzyme
12 microL water

This digestion was then run on a gel for 1 hour.

6 colonies of the re-transformed plasmids were put in LB and ampicillin and incubated over night at 37 degrees, shaking.

9/12/2009 -

DNA concentration of the 6 colonies (in ng/microL): -

1) 10.1
2) 6.3
3) 17.9
4) 10.6
5) 9.0
6) 14.0

These concentrations are too low to carry out a digestion.

9/12/2009 -

I met with Tracey to discuss how best to set out my report. We discussed how to set each part of the results into sections and to try and outline the project as soon as possible so results can be fitted in as we do the experiments.

Over the christmas holidays I will write up the introduction and begin the results using those we have collected already. I will also set out the outline for the rest of the report.