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Miniprep of pSB1C3-LovTAP, pSB1C3-RFP and pCEP4-Readout
Protocol: Miniprep
The slim tubes can be centrifuged in the machine in front of the "Gel hood", at 4000 rpm for 10 min. The fatter ones, in the E. coli centrifuge by the fridge (the tip can be left inside, since it floats).
Pellets resuspended with RNase containing buffer (Resuspension Buffer R3, from Invitrogen, equivalent to Buffer P1 from Qiagen, in Sowmya's box in the fridge). Note: keep the buffer in ice if you are not bringing it back to the fridge for some minutes.
We then use the QIAGEN QIAprep Spin Miniprep Kit with their [http://www.qiagen.com/literature/render.aspx?id=370 protocol] (page 22) and a microcentrifuge.
Minipreps made for yesterday's overnight cultures.
DNA Concentration Measurement for the Minipreps
Protocol: DNA Concentration Measurement
- Take a 6 µl aliquote of the DNA and put back the main DNA tube in the fridge.
- Go to the room by the E.Coli lab (LBTM, not on Friday morning!) with:
- The 6 µl aliquote
- A 10 µl pipet
- Optionally, the buffer you used for DNA elution (there might be some next to the machine).
- The machine is the NanoDrop Spectrophotometer.
- On the computer, click on "Nucleic Acid".
- Put a 2 µl drop of (nuclease-free) water on the machine's tip as you are asked to and measure.
- Clean tips (both sides) with a quarter of tissue.
- Add 2 µl of the buffer you use and click on "Blank".
- Clean tips (both sides).
- Add 2 µl of your DNA sample and click "Measure".
- Clean tips (both sides) with a tissue.
- Take 2 measurements per sample (for averaging).
- Print the report when you are done
- Click on exit.
The important numbers are:
- 260/280 ratio, must be > 1.8
- 260/230 ratio, must be > 2 (too big, > 2.5? , might mean too much salts)
- Of course the DNA concentration.
A Nanodrop measurement of the minipreps was performed. On average, it yielded acceptable concentrations at around 100 ng/µl. This chloramphenicol concentration was apparently sufficient.
We now have minipreps of the LovTAP BioBrick that can be shipped, minipreps of pCEP4-HA-RO that should be checked by PCR before a Maxiprep can be done, and a reserve of pSB1C3-RFP plasmid for our other BioBricks.
CHO Cells Transfection with Melanopsin and LovTAP
Protocol: Transfection of CHO cells
This is the transfection protocol used at the LBTC lab for CHO DG44 cells. The transfection reagent is PEI ([http://en.wikipedia.org/wiki/Polyethylenimine polyethylenimine]).
Please use the provided Excel sheet to calculate the volume of plasmid you should add to the cells. Replace every value in red by your own, then print the sheet out and follow the provided protocol.
1. Passage seed 1 day prior to transfection.
2. Prepare tubes (yellow caps with holes) by addition of the calculated amount of DNA.
3. Centrifuge the necessary volume of seed (after a PCV measurement), remove conditioned medium with the pump (use a 2 ml serological pipet with a broken neck) and resuspend (first in 10 ml) in necessary volume of fresh medium to achieve the required cell density (3 mio/ml).
4. Add 5 mL of the cell suspension to the tube with DNA and mix orbitally.
5. Add the PEI (45 µl) to the Cell+DNA mixture as soon as possible, flick 3 times.
6. Place in the incubator at 37°C.
CHO cells from a seed culture have been transfected. We made two tubes with 100% LovTAP and two tubes with 50% LovTAP and 50% filler DNA. They were later used for a Western blot. These cells were transfected in the morning and were put in the incubator at 11.20. No readout plasmid was used as it was still not ready at the time.
Then we transfected another batch of CHO cells, this time with melanopsin and its pGL-GFP readout. We made two tubes that were transfected with 50% GFP and 50% melanopsin, and two others that were transfected with 50% GFP and 50% filler so we can get a pGL-GFP leakage baseline.
Culture setup in blue light
A program was written and uploaded to the Arduino in order to set constant illumination for all LEDs. The melanopsin-transfected cells have been loaded in a 24-well plate (three hours post-transfection), along with the GFP ones and a sample of seed cells. The intent is to measure fluorescence with the Guava machine regularly, every few hours.
Fluorescence measurement
Protocol: Fluorescence (Guava)
Prepare your samples by measuring their PCV (or estimating the cell amount according to the doubling rate). Dilute them with PBS in order to have between 200 and 500 cells/µl. Prepare at least one well that has seed cells.
Steps 1 to 4 are optional and should be done from time to time.
1. Trash the waste on the bottom right of the machine if it is full before you start.
2. Put tubes with bleach (detergent) at the right positions.
3. Run 'Cytosoft 5.3'
4. Click Clean and Shut Down -> This would take around 15 min.
5. After cleaning, click Guava Express Plus on the left column.
6. Go to Analysis mode and click 'Open Data Set'.
7. Go to the 'iGEM' folder and open the 'Setting' file.
8. Go to Acquisition - and hold here.
9. Go to the desktop and run WorkEdit 5.3.
10. Highlight the wells you are going to use, check "Acquire this sample".
11. Label them as Guava Express Plus, check the "Mix for 3 seconds", set the speed from high to medium. Optionally, fill the sample ID and the dilution factor.
12. Save and go back to Cytosoft 5.3.
13. Go to Acquisition, start the worklist.
14. Place the 96-well plate into the tray, make sure the A1 well is where it should be.
15. Name the file as 'Today's date_title'
16. When you are asked to adjust the settings, check a well that contains seed cells.
17. Compare with the worklist, check if the flow and the amount of cells detected are reasonable.
18. Click "Next step" and then "Resume".
19. Wait until all the wells are measured - data will be saved automatically.
20. Take the 96-well plate out and insert the tubes that are required for cleaning.
21. Go to Main menu and click 'Clean and shut down'.
A first sample was taken and measured as the cells were put into the shaker.
- The times were as follows:
- 16:00 Transfection
- 19:00 Put tube spin cells in well plate (3h after transfection). Take T = 0h sample of first well plate transfer
- 22:00 Take T = 3h sample of first well plate transfer
