User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/06/02

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χρόνος πέρασμα June 2nd 2011


  • Experiment that proves if the digestion linearized pBBRMCS5 succesful. Ligation and transformation of the digestion to see if the digestion was performed as we want it, with both HindIII and XbaI acting upon the same plasmid.

  • Today I checked if the petri dishes I left incubating grew bacteria. In brief, the petri plate that was inoculated with bacteria transformed with the digested plasmid should not produce colonies, as the plasmid is no longer circular and cannot replicate to confer the bacteria that carries it resistance to gentamicin. The good news is that there were indeed no colonies =), this means the digestion was succesful in linearizing the plasmid. The problem here is that we are not sure if BOTH restriction enzymes acted in the same plasmid or if only one of them did, as both scenarios would give the same result, no bacteria growing in the solid medium with gentamicin.
  • Melisa Rivas and I were supposed to continue with the experimental design, filling the tips of the plasmid/blunt the ends; but Miguel Ramírez made us aware of the digestion uncertainty. The ligation was performed with the following reactant concentrations:

Tube 1 Control tube
5.5 µl 8 µl H20
1 µl 1 µl Buffer 10x with dNTPs
2.5 µl 0 µl DNA (plasmid)
1 µl 1 µl T4 Ligase
10 µl 10 µl Total

  • In theory, the ligation should be left incubating at 25°C during 12 hours or even overnight, but we left it approximately six hours as Miguel told us this should be enough. If the digestion was performed correctly, meaning most of the plasmid (if not all) were cut in both restriction sites, the sticky ends that were produced at the ends of the linearized plasmid wouldn't be compatible to be ligated and the ligation procedure should be virtually impossible. In the other hand, if the plasmids were cut just by one enzyme, then, the sticky ends left should be completely compatible and the ligation reaction should be performed without problems, resulting in circular pBBRMCS5 (just as the one freshly extracted). At 6:00 pm I transformed competent DH5α with the ligations (three tubes, one with the "ligated" plasmid, one with the ligation control and one last as a transformation control) following the protocol Miguel gave us. To prepare the petri plates I used solid medium that was stored in our laboratory, I used 120 ml of solid medium LB with 120 µl of gentamicin. I made the mistake of taking too long to pour the medium in the plates, as the medium was already solidificating when I started... as a consequence, the petri dishes resulted more ugly than the uglyness (and with more bubbles than a can of coke agitated for one hour). The protocol says that I have to put 200 µl of the transformed bacteria in the petri plates, Fabricio López told me that I could put less (~100 µl) if I centrifugate first the transformations and take directy from the pellet the bacteria.
  • In total I made four petri plates:
  1)With the transformation with the ligated plasmid. If well digested, no colonies expected.
  2)With the transformation with the ligation control. No plasmid added to the ligation reaction, no plasmid to transform, no colonies expected.
  3)With the transformation control. No plasmid used during the transformation, no colonies expected.
  4)The plating control. No bacteria added to the plate, no colonies expected.