Team:EPF-Lausanne/Notebook/1 August 2012

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Contents

Morning : Miniprep of pHY42 transformed colonies

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 protocol (page 22) and a microcentrifuge.


Nanodrop results :

Sample 260/280 260/230 conc. ng/µL
1 1.84 2.53 481.5
2 1.85 2.52 425.4
3 1.85 2.56 420.2
4 1.84 2.53 398.9
Comments

The concentration values are an average of two readings. The DNA quality is acceptable.

Afternoon : Digestion of all the plasmids for testing and ligation

Note: the results of this digestion were completely screwed up, though subsequent digestions of the same plasmids were OK. There's a bug in here!

We did 5 digestions:

PHY42 plasmid (melanopsin) x 4 samples

In one tube:

  • Water: 169.2 µl
  • N4 buffer x10: 20 µl
  • BSA x100: 2 µl
  • ApaI (10 u/µl): 2 µl

Then, in each one of 4 tubes (one per miniprep):

  • 48.3 µl of the previous mix
  • 1.2 µl of DNA
  • Let 30 minutes at RT (26ºC)
  • Add 0.5 µl of NheI (10 u/µl)
  • Let 2 hours at 37ºC

The enzyme cut out the melanopsin from the pcDNA3.1 backbone, so we know we should have a 5413 bp band, and the other with the size of the melanopsin gene.

pMP for control gel x 3 samples

Using enzymes cutting the plasmid into 3655 and 1026 bp parts. In one tube:

  • Water: 121.5 µl
  • N4 buffer x10: 15 µl
  • BSA x100: 1.5 µl
  • HindIII-HF (20 u/µl): 1.5 µl
  • XbaI (20 u/µl): 1.5 µl

Then, in each one of 3 tubes (one per miniprep):

  • 47 µl of the previous mix
  • 3 µl of DNA
  • Let digest at 37ºC for 2 hours

pMP for ligation x 3

Using enzymes cutting the plasmid for subsequent ligation In one tube:

  • Water: 366.5 µl
  • N2 buffer x10: 45 µl
  • BSA x100: 4.5 µl
  • SpeI (10 u/µl): 2 µl
  • NotI (10 u/µl): 5 µl (more because it's non-optimal in buffer N2)

Then, in each one of 3 tubes (one per miniprep):

  • 141 µl of the previous mix
  • 9 µl of DNA
  • Let digest at 37ºC for 2 hours

pGL for ligation x 2

Using enzymes cutting the plasmid for subsequent ligation with TNFR and EGFP. In one tube:

  • Water: 366.5 µl
  • N2 buffer x10: 45 µl
  • BSA x100: 4.5 µl
  • SpeI (10 u/µl): 2 µl
  • NotI (10 u/µl): 5 µl (more because it's non-optimal in buffer N2)

Then, in each one of 3 tubes (one per miniprep):

  • 141 µl of the previous mix
  • 9 µl of DNA
  • Let digest at 37ºC for 2 hours

pGL for ligation x 1

Using enzymes cutting the plasmid for subsequent ligation with SEAP. In one tube:

  • Water: 256.5 µl
  • N4 buffer x10: 30 µl
  • HindIII-HF (20 u/µl): 1 µl
  • FseI (2 u/µl): 15 µl
  • 12 µl of DNA
  • Let digest at 37ºC for 2 hours


Protocol: Restriction site digestion


  1. Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
    1. NEBcutter for finding cutting enzymes.
    2. 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.



Comments
By mistake, we prepared everything under the assumption that each miniprep had 15 µl of solution, instead of the 50 µl it actually has.


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