User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/03

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Starting again with the standardization :D

ABSTRACT

  • Plasmid pBBRMCS5 isolation to start over the standardization, following Orlando´s recommendations. Because of the isolation failed we left growing bacteria to start again.

  • To explain what made us decide to start again i´m going to reference the words of my team partner Daniel:


Paulina Alatriste, Pablo García and I planned to put to digest again the digestion with the same 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.


  • Having done this we decided to start again with the process, and the first step was to isolate pBBRMCS5 plasmid from E. coli cells. We followed the protocol provided by Miguel except the steps: .... Unfortunately we could not extract plasmid, no pellet was observed at the final step after the treatment with all the solutions. The first possible explanation is that we didn´t follow the protocol correctly, the second explanation is that in some way the cultures used affect the extraction process. This is because we used bacteria that have been growing more than one day in 5 ml of liquid medium. We think that the protocol is designed for a certain concentration of cells, and that too much could diminished the reliability of the extraction.


  • The only thing left to do was to grow bacteria in liquid medium and Daniel is going to extract plasmid tomorrow.


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