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Ligation plates observation
Colonies were observed on the pCEP4-HA-RO plate transformed the evening before. The pCEP4-HA control was fairly clean. The pCEP4-RO (without the [http://en.wikipedia.org/wiki/HA-tag HA tag]) plate had few colonies, and a lot of them had grown on the control, so we decided to keep only the pCEP4-HA-RO one. We used the plate for a colony PCR on the following day.
Glycerol stock
Made a pHY42 bacteria glycerol stock.
New LB
Protocol: Preparation of LB Medium
Put 25g of powder in 400ml + add ~100ml to complete to 500ml. Put in autoclave (don't forget to loosen the caps!).
Made 2l of LB.
New CHO seed line
Protocol: Cell Passaging
These are the general mammalian cell passaging rules, they work for CHO cells and HEK cells.
The cells should be left to grow for two days, after what their density should be reduced and the medium should be changed.
We usually bring them either to 2 mio/ml, either to 1 mio/ml.
- Put the volume of medium that corresponds to the amount of cells you'd like to passage at 37°C, leave it there to heat for 10 minutes (more for bigger volumes, less for smaller).
- Measure the PCV of your current cell sample. Usually, the cells double every day, so you can estimate their number, but a PCV is more precise. Take 200 µl of cell suspension in a mini-PCV tube, centrifuge them (1 min at 5000 rpm), the cells will fall down in the thin tube at the bottom. Use the VoluPAC Reader to measure the PCV (the volume in µl the centrifuged cells take).
- If you're dealing with HEK cells, the PCV is also the number of millions of cells/ml you have.
- If you have CHO cells, divide the PCV by 0.4 to obtain the mio/ml cell density.
- Take the volume that will contain the amount of cells you need from the original sample in a centrifuge tube, centrifuge it at 1500 rpm for 3 minutes. A pellet will form.
- Remove the supernatant (under the hood) either with the vacuum pump, either by simply discarding it.
- Resuspend in 10 ml of warmed medium (pipet up and down)
- Add the remaining medium, mix again
- Split the cultures into 10 ml samples in separate tubes, put them in the shaker at 37°C, passage again after 2 days.
Passaged CHO seed that will be used for WB and for our transfections.
PCR cleanup
Protocol: PCR Cleanup
After doing a PCR, the resulting DNA should be cleaned up to get
rid of the primers, polymerases, dNTPs and the various other reagents
used in the PCR. This can also be used to remove small fragments
of DNA from other sources (such as digestions).
We used Macherey-Nagel's "Nucleospin® Gel and PCR clean-up" kit. The manual can be found here: [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf Gel and PCR clean-up Manual]
The kit uses a silica membrane to bind DNA which is then washed with several different buffers. The final step is the removal from the membrane by elution and recovery of our cleaned DNA.
Note: To increase the yield we applied the optional steps in 4 and 5 in the PCR cleanup protocol on pages 18 and 19. By incubating the columns at 70 degrees and eluting the DNA with heated elution buffer the yield of longer fragments can be increased.
The LovTAP PCR products (flanked with BB prefix and suffix) were all mixed together (these were different tubes which gave the same bands) and run through a Macherey Nagel PCR cleanup kit.
Nanodrop of the cleaned LovTAP BioBrick
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.
The Nanodrop showed a very low DNA yield, slightly more than 10 ng/µl.
Digestions
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 (from the Spring iGEM Distribution, linearized backbone in a tube) and the purified LovTAP PCR fragment were digested with EcoRI and PstI. The following mixes were made:
- LovTAP-BB
- H20: 28 µl
- N2 buffer: 5 µl
- BSA 10x: 5 µl
- EcoRI-HF: 1 µl
- PstI: 1 µl
- LovTAP-BB DNA: 10 µl
- pSB1C3
- H20: 34 µl
- N2 buffer: 5 µl
- BSA 10x: 5 µl
- EcoRI-HF: 1 µl
- PstI: 1 µl
- pSB1C3: 4 µl
They were incubated at 37°C from 19.50 to 20.50.
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).
Finally, for the insertion of LovTAP into pSB1C3, a ligation was performed.
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)
The ligation products (pSB1C3-LovTAP) and a non-linearized pSB1C3-RFP (from the iGEM dry DNA plates, transformed to have more of this backbone) were used to transform bacteria. They were cultured on chloramphenicol plates!
The LovTAP bacteria were used for a colony PCR on the following day.
Maxiprep culture for everything
Protocol: MaxiPrep
The evening before, take a big Erlenmeyer (at least 1L) and put 200ml LB in it. Add the appropriate antibiotics at the correct concentration (ampicilin: 200ul of 100mg/ml solution). Put in bacteria from a single colony of a freshly streaked plate or from a glycerol stock (warning: taking bacteria from glycerol stock seems to cause them to start growing later - due to thawing? - add one-two hours to the incubation time). Put them in the incubator for 14-15 hours (the contents of the bottle should be yellow-ish between translucid and opaque).
We then use the MaxiPrep kit (Plasmid DNA Purification kit) and protocol from Macherey-Nagel.
The complete handbook can be found [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NuBo.pdf here]. We usually use the protocol that starts at page 24 for "Maxi".
The glycerol stock made on the day before was used to start a maxiprep culture of pHY42, pcDNA3.1(+)-LovTAP and pGL-GFP in an erlenmeyer, overnight.
Soldering
The assembly of our bioreactor plates, their LEDs and the Arduino was started.
