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LovTAP Readout Biobrick PCR
Protocol: PCR
PCR is a reaction that makes it possible (and relatively easy) to amplify
a certain region of DNA. The first step is the selection of that region
(and the design of the relevant primers). Primer design can be done by hand, or by
using our Primer Design Helper. Once
done, order the primers (in our case, we ordered from them [http://www.idtdna.com/ IDT]).
When you've received the primers, prepare them and make sure you've got your PCR kit (we used the "Phusion® High-Fidelity DNA Polymerase"). Start preparing your master mix, the composition for one tube is:
1X Mastermix 20μl reaction, add in this order
| Reagent | Volume [μl] |
|---|---|
| Water | Complete to total volume of 20μl |
| HF-Buffer (5x) | 4 |
| DMSO (optional) | 0.6 |
| dNTPs | 0.4 |
| Forward primer (50μM) | 0.2 |
| Reverse primer (50μM) | 0.2 |
| Template (10ng/μl) | 0.5 |
| Phusion HF polymerase | 0.2 |
Prepare one or two extra tubes-worth of reagent (you'll use some liquid on the walls of your tips).
Once you've finished, you should run the resulting products on a gel to check if everything went as planned.
Tips
- Thaw the HF-Buffer, DMSO and dNTPs before making the mastermix.
- Avoid taking the Phusion-HF polymerase out of the freezer (only take it out briefly when you need to add it).
- If the reactions have different primers and/or template, add the polymerase right after the dNTPs, split the mastermix and add the rest.
- Don't forget positive and negative controls
- Primers should have similar Tms (less than 5°C).
- Primer Tm calculation is a less exact science than it should be (just test several tools and compare their results). If you're not sure what the correct Tm is, consider using a gradient PCR.
- Avoid primers with strong secondary structures.
- PCR can introduce mutations. Don't forget to sequence your final product (this could be your final plasmid): you really don't want to lose a few weeks because of a "corrupt" plasmid.
A gradient PCR was run with the appropriate Biobricking primers, with a GFP positive control. The PCR products were run on a gel.
The expected product size was 1273 bp, which is what we observe for all lanes of the gel. This version of the BioBricking primers finally seems to work, at all temperatures.
pSB1C3 and NFAT BioBrick Digestion
Protocol: Restriction site digestion
- Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
- [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes.
- [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double Digest Finder] for the parameters.
- Calculate the amounts required of:
- DNA
- Buffer (usually from 10x to 1x)
- BSA, if needed (usually from 100x to 1x)
- Enzymes (depends on the amount of DNA)
- Water
- Get the recommended buffer (and BSA if needed) from the freezer and let defreeze.
- Mix all the ingredients, except DNA, in a tube.
- Note: Enzymes should stay no longer than a couple of minutes out of the freezer. Don't touch the bottom of the tubes! Don't vortex!
- Distribute the mix in as many tubes as DNA samples and add the DNA.
- Keep in the Thermomixer at the recommended temperature.
Sowmya's recommended amounts (50 µl total solution):
- 5 µl of 10x buffer
- 0.5 µl of 100x BSA
- 1 µl of each enzyme
- 5 µl of DNA
- 37.5 (up to 50 µl) of water.
Protocol based on what was done on July the 4th.
pSB1C3-NFAT Ligation
Protocol: Ligation
Ligation is a method of combining several DNA fragments into a single plasmid. This is often the
step following a PCR (and a PCR cleanup) or a gel extraction. You can also do a "dirty" ligation, where you follow a certain number of digestions directly by a ligation.
- Download the following spreadsheet : File:Team-EPF-Lausanne Ligation.xls
- Fill in the pink areas with the vector and fragment concentration, their size and the ratio.
- Add all the suggested ingredients order in a microcentrifuge tube, in the order they appear.
- Ligate for 2 hours at 14ºC.
- Immediately transform competent bacteria with the ligation product.
Note: This protocol hasn't been optimized for blunt-end ligation (though it might still work).
The NFAT BioBricked PCR product was digested with EcoRI and PstI, and so was the linearized backbone received from the iGEM Headquarters. We ligated them together, following our standard protocol.
pSB1C3-NFAT Transformation
Protocol: E.Coli Transformation
- Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
- As soon as it is thawed, add 50µl of the cells to the DNA (~50-100 ng of pure plasmid, or some 2 µl usually)
- Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
- Put the tube with DNA+E.coli at 42°C for 45 sec - 1 min (heat shock)
- Add 400 µl of LB broth and place at 37°C for 20-30 min (shaking)
- Spread the cells on the prewarmed plate (and let it dry)
- Incubate the plate upside-down at 37°C for ~14-15 hours (leaving it more than 16h decreases the plasmid quality)
A transformation of the ligated pSB1C3-NFAT product was performed, and the plates were left overnight, as usual.
Transfection of pcDNA3.1(+)-LovTAP into CHO Cells
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.
Using our usual protocol and the Excel provided in it, we transfected LovTAP and its readout into CHO DG44 cells. We made two tubes containing 50% LovTAP and 50% of its dsRed readout, two tubes containing 50% LovTAP and 50% filler DNA, and two tubes containing 50% readout and 50% filler.
They were made for a flow cytometry analysis at the FACS facility at EPFL.
Flow Cytometry for Detection of Red Fluorescence
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 test for a possible detection of dsRed with the Guava machine in our lab was performed. The Guava is not very well-adapted for the detection of red fluorescence, though. We managed to see a red fluorescence difference between the cells that were transfected with dsRed and the ones that weren't, though, so we assume the transfection worked.
