Team:EPF-Lausanne/Notebook/11 September 2012

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Contents

BioBrick digestions

Protocol: Restriction site digestion


  1. Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
    1. [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes.
    2. [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double Digest Finder] for the parameters.
  2. Calculate the amounts required of:
    1. DNA
    2. Buffer (usually from 10x to 1x)
    3. BSA, if needed (usually from 100x to 1x)
    4. Enzymes (depends on the amount of DNA)
    5. Water
  3. Get the recommended buffer (and BSA if needed) from the freezer and let defreeze.
  4. Mix all the ingredients, except DNA, in a tube.
  5. 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!
  6. Distribute the mix in as many tubes as DNA samples and add the DNA.
  7. 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.


The linearized pSB1C3 and the melanopsin BioBricked PCR amplicon were both digested with NotI. We also ran a digestion of the circular pSB1C3 and of the melanopsin amplicon with EcoRI and SpeI, in case the first one didn't work out (it's a digestion on the very ends of a linearized backbone after all).

Ligation of melanopsin into pSB1C3

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.

  1. Download the following spreadsheet : File:Team-EPF-Lausanne Ligation.xls
  2. Fill in the pink areas with the vector and fragment concentration, their size and the ratio.
  3. Add all the suggested ingredients order in a microcentrifuge tube, in the order they appear.
  4. Ligate for 2 hours at 14ºC.
  5. Immediately transform competent bacteria with the ligation product.

Note: This protocol hasn't been optimized for blunt-end ligation (though it might still work).

  • Ligation of digested pSB1C3 with the digested melanopsin PCR amplicon (NotI).
  • Ligation of pSB1C3 with the melanopsin PCR amplicon (EcoRI and SpeI).

The ligation result should be the same.


Maxiprep of pCEP4-RO

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".


Yesterday's maxiprep failed (no pellet). Started another maxiprep of the Read-out plasmid.