χρόνος πέρασμα June 3rd 2011
- The experiment to check if pBBRMCS5 was digested with HindIII and XbaI failed. Plasmid pBBRMCS5 extraction to start over the standarization failed. Bacteria were left growing to try again.
- Today I checked the petri dishes I inoculated yesterday. Besides the fact the plates were very irregular (this difficults the colony observation), the main plate did have bacteria growing; the one that was plated with E. coli strain DH5α that was transformed with the allegedly doubled digested plasmid pBBRMCS5. Unfortunately, I did not make another petri variant, a petri dish that should have been inoculated with transformed bacteria whose plasmid digestion should have been performed with only one restriction enzyme in order to see the colony ratio among these two experiments. Saaadneeeess...I failed.
- Paulina Alatriste, Pablo García and I planned to put to digeste again the digestion with the samen enzyme concentration another day hoping that this would finally make all plasmids to be double digested. We went to see Miguel but he was absent, fortunately Orlando was there to help us, he told us that the restriction enzyme action efficiency is sometimes diminished once a DNA molecule is linearized. We checked if our enzymes, HindIII and XbaI were affected by this and saw that they did not (efficiency to cut linear DNA of 99% and 90% respectively). He also told us that if the concentration we used in the first digestion did not prove to be efficient left once overnight, there should not be any different outcome doing it again, wheter one of the enzymes isn't working properly or we aren't using the adecuate enzyme concentration. Another scenario might be happening: once both enzymes cut in their respectives sites, a very little piece of DNA is released from the plasmid (this fragment cannot be seen in an electrophoresis gel because of its size) and remains in the plasmid solution that is put to be ligated. There's this chance that the ligation occurs with this fragment and that is how colonies can be observed (I thought the probabilities this could happend should be minimal but apparently this did happen frequently because of the number of colonies that were observed). Orlando recommends us to purify the plasmid from the electrophoresis gel itself in order to make sure that this fragment is not been integrated again into the plasmid.
- We decided to start it over again and do Orlando's proposition. There were bacteria with the plasmid pBBRMCS5 growing in liquid medium ready to be extracted. Paulina, Pablo and I followed the protocol provided by Miguel with the same exceptions done here. We don't know why but there was no plasmid isolated with the protocol (no plasmid "pellet" was observed after the treatment with the solutions). There might be two possible explanaitions: One is that we did not follow the protocol correctly, the other is that it might not be adecuate to use bacteria that have been growing more than one day in 5 ml of liquid medium. The test tubes in which the bacteria grew that we used had been left incubating for too long, the solutions quantities that come in the protocol should not be enough for so many bacteria. But this is only a speculation.
- There was nothing left to do besides leaving bacteria to grow again in liquid medium. I will extract the plasmid tomorrow using the protocol.
- Fernando Montaño, one of our advisors, helped me complete the form needed to release the Pam Silver's package once it arrives to Mexico. As Miguel was absent, I didn't deliver it signed by him to Maricarmen (the one in charge with all the mexican customs things).