Purification of DNA and Plasmid

06/11/09 -

Attended Lab meeting where I was able to update Dylan with the progress my project is making.

Cells were passaged:
P.34 and P.35 were given medium

10/11/09 -

7 flasks of my P.29 cells became infected and were thrown away.

13/11/09 -

Whilst carrying out her own experiments Tracey tested the expression of Zmpste24 in SW1353 cells and found it was highly expressed. Therefore we decided to extract and purify the Zmpste24 from these cells using the following method:

- Add 150 microL to 50 microL of the PCR product
- Add 200 microL of phenol cholorform
- Vortex for 5 seconds
- Centrifuge at 13,000 rpm for 10 minutes at 4 degrees Celsius
- Put the upper phase into a new eppendorf
- Add 500 microL 100% ethanol
- Add 20 microL sodium acetate
- Add 1 microL glycogen (this will act as a branching molecule which will grab the smaller fragments of DNA and bring them down into the pellet)
- Centrifuge at 13,000 rpm for 10 minutes at 4 degrees celcius
- Remove supernatant
- Add 500 microL 70% ethanol (do not resuspend)
- Spin as before
- Remove supernatant
- Leave to air dry (will take about 10 minutes if all dregs are removed)
- Resuspend in 100 microL water (as there was a big pellet)

The DNA was then stored at -20 degrees Celsius.

I met with Dylan to discuss my project and the next steps we will be taking. The first issue that needed to be addressed was whether to keep all my flasks of cells as I have too many. It was decided to keep P.35 and P.34 to extract the RNA at a later date, then keep at most two flasks of P.29 to keep passaging, harvesting RNA and Protein each time, keeping the flasks of cells to a maxium of 2. The rest could be thrown away. The cells will need to be split at a certain density so that we know how many population doublings have occurred in each passage, I will work with Alba to do this.

Over the next week or so I will also be working with Caroline Pennington to quantify the amount of Zmpste24 in the RNA I have already harvested and will also be looking at the amount of mir-34 and Zmpste24 in a number of cancer cell lines.

16/11/09 -

The purifyied Zmpste24 DNA was digested with the restriction enzyme HindIII to create sticky ends using the following method: -

Into an eppendorf tube add:-
- 80 microL PCR product (purified DNA from previous step)
- 10 microL HindIII buffer
- 10 microL HindIII enzyme

Incubate at 37 degrees Celsius for 3 hours.

I then harvested RNA from P.28 and P.29 cells using the same method as previously.

RNA concentration:

P.28 = 465 ng/microL (260/280 = 1.95, 260/230 = 1.26)
P.29 = 475 ng/microL (260/280 = 1.92, 260/230 = 1.28)

17/11/09 -

The digested DNA was then purified again using the phenol cholorform method.

100 microL of water was added to 100 microL of the product then the method was followed exactly as before.
The pellet was resuspended in 50 microL water.

The purified product was then run on a 1.5% agarose gel (made as before) to check if anything had been recovered. But as the gel was setting it had leaked making the gel very thin. After looking under UV light it no band could be seen.

The product was run again along with a 1kb + ladder and pmir report which will be used as the vector. This time a clear band could be seen.

19/11/09 -

All of my cells, except for two flask of P.29, became infected and had to be thrown away. It is thought to be because the medium contained no antibiotics.

Pen Strep antibiotics were added to my medium, then one flask of P.29 cells was passaged to two P.30 flasks. The cells from the other flask were harvested to extract protein as follows:

- Wash cells with 1 x PBS
- Add 1 ml PBS
- Scrape
- Transfer to an eppendorf tube
- Spin at 13,000 rpm for 5 minutes at 4 degrees Celsius
- Remove PBS
- Place pellet on ice
- Resuspend in RIPA buffer (use 3 x the volume of the pellet, in this case we used 100 microL)
(Ensure RIPA buffer has had a protease inhibitor tablet dissolved into it first)
- Leave on ice for 20 minutes
- Centrifuge at 10,000 rpm for 10 minutes at 4 degress Celsius
- Transfer supernatant to a new eppendorf
- Store at -20 degrees Celsius

An agarose gel containing the linearised plasmid was viewed under UV light to allow visualisation of the plasmid, which was then cut out of the gel and put into an eppendorf tube. The plasmid was then extracted using a QIAGEN extraction kit.

20/11/09 -

The DNA concentration of the Zmpste24 PCR product and the pmir report was measured using the nanodrop:

DNA concentration:
Pmir Reporter (which has been cut with HindIII) = 7.6 ng/micro L
Zmpste24 PCR product = 35.6 ng/microL

Four ligations were then set up to run over night.

The amount of insert was kept constant and a final volume of 20 microL was made up using water. The ligations were set up as follows:

1) 1 microL vector + 1 microL insert + 2 microL ligase + 2 microL buffer + 14 microL water
2) 5 microL vector + 1 microL insert + 2 microL ligase + 2 microL buffer + 10 microL water
3) 1 microL vector + 2 microL ligase + 2 microL buffer + 15 microL water
4) 5 microL vector + 2 microL ligase + 2 microL buffer + 11 microL water

Ligations 3 and 4 will act as controls.

The ligations were incubated at 16 degrees Celsius over night in the PCR machine.