The Taqman results gave a circumstantial relationship between miR-34 and Zmpste24, as it was seen in cells high in miR-34 there was a low level of Zmpste24 and vice versa, indicating that Zmpste24 was a likely target for miR-34.
10 more cultures were mini-prepped and digested using the Sac1 restriction enzyme. The gel image again revealed the insert had been incorporated in the wrong way. But every vector had the insert, which is unusual due to the low efficiency rate of the transformation so it is thought that the undigested vector is predominant on the plate.
A transfection was set up using the SW1353 cells which had been plated and were allowed to grow until they were confluent. The cells were tested with a control (scrambled) which scrambles the miR-34 binding site and a miR-34 mimic at 3 different concentrations, 10nM, 20nM, and 50nM.
The following volumes were added to 200 microL serum free medium in separate eppendorfs
Control:
10nM - 0.4 microL
20nM - 0.8 microL
50nM - 2 microL
miR-34 mimic
10nM - 1 microL
20nM - 2 microL
50nM - 5 microL
The eppendorfs were then incubated at room temperature for 5 minutes.
During this incubation period a 15x mastermix of lipofectamine 2000 (a transfection agent which, when combined with the mimic, will transport the mimic into the cell) and serum free medium was made.
1X = 4 microL lipofectamine, 196 microL serum free medium
15X = 60 microL lipofectamine, 2.9mls serum free medium
Once the incubation period was over 200 microL of the mastermix was added to each eppendorf. These were then incubated at room temperature for 20 minutes.
After this incubation period was finished the solutions were added to each well in a drop-wise fashion. The plate was then incubated at 37 degrees Celsius for 48 hours. How the 6 well plate was organised is shown below.
21/01/2010 -
A PCR purification was done using a QIAgen QIAquick PCR purification as per the protocol provided with the following changes:
100microL PB1 added to sample
50microL water used to elute DNA rather than EB
The DNA was then digested using 10microL H3 enzyme, 10microL H3 buffer, 30microL water (this brings the final volume to 100microL). The sample was then digested for 3 hours at 37 degrees Celsius.
5 microL of the sample was run on a 1.5% agarose gel to test the digestion. The digestion worked very well and the insert and vector were cleanly cut. Again the insert was 400bp, but this time no background vector appeared on the gel image.
25/01/2010
A western blot was set up using the transfected cells. The total protein concentration was measured and the following volumes were loaded (+4 microL loading buffer) to ensure the same amount of protein was loaded in each lane (80nM)
Transfection Absorbance Protein Conc. (ng/microL) Volume loaded (microL)
Scrambled10 0.48 4.8 16.6
Scrambled 20 0.65 6.4 12.5
Scrambled 50 0.57 5.7 14
miR-34 10 0.63 6.3 12.7
miR-34 20 0.51 5.1 9.8
miR-34 50 0.52 5.2 5.1
The protein and loading buffer were boiled for 5 minutes at 90 degrees Celsius on a hot block before loading. The gel was then run at 150V for 1 hour.
Once the gel has finished running it was transferred to a membrane using the same method as previously described.
While the proteins were being transferred to the membrane 12 more colonies were picked and incubated in LB + 5 microL ampicillin and incubated overnight, shaking, at 37 degrees Celsius.
26/01/2010
The 'quick and dirty' mini-prep method was used to purify the plasmid DNA.
The primary Zmpste24 antibody was applied to the membrane yesterday and it was left in the cold room overnight.
The secondary antibody was then applied and the membrane put on the shaking platform for 1 hour.
After this hour it was washed in PBS tween, 2 x 5 mins on the shaking platform. Then 1ml of a chemiluminescent was added and left to incubate for 1 minute. The membrane was then transferred to an AUTO-RAD sheet and left to develop for 1, 5 and 10 minutes.
The developed AUTO-RAD sheets can be seen in my lab diary.
The plasmid DNA was digested with Sac1 as previously and the samples run on an electrophoresis gel. Only one vector had incorporated the insert, but again in the wrong orientation.
28/1/2010
A flask of P.34 cells were harvested and protein and RNA was extracted using the same method as previously described. New medium was added to the second flask to keep the cells alive until we can photograph them. These cells are now reaching the stage of senescence.
