User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/05/27

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χρόνος πέρασμα May 27th 2011

  • Today, Melisa Rivas and I started following the plasmid extraction protocol with the Roche's High Pure Plasmid Isolation Kit to isolate the plasmid pBBRMCS5.
  • I made an awful mistake, I didn't resuspend the bacteria pellet before I added the solution #2 lysis buffer; according to the manufacturer (and also according to any experienced person) once this solution is added, you must treat the bacteria lysis very carefully, as we do not want the chromosomal DNA to break apart. Nonetheless, I followed the procedure step by step and obtained 4 eppendorf tubes with 40 µl of the "isolated" plasmid.
  • I made two modifications to Roche's protocol: a) I didn't do step #6; the protocol says it must be done if the E.coli strain has high nuclease activity. The strain DH5α has low nuclease activity. Still Fabricio López told me to do this washing step next time. b) I used 40 µl of the elution buffer instead of the 100 µl that says in the protocol; I did this because we want the plasmidic DNA very concentrated.
  • I ran an gel electrophoresis to see if I could still see the isolated plasmids even with my mistake. In this gel I put my extraction as well as the extraction Fabricio López did yesterday (very unfortunately he told me he did the very same mistake). I stained the gel with 200 µl of ethidium bromide, as I used 200 ml of destilled water (the ethidium bromide concentration must be 1 µl per 1 ml of solvent).
  • The gel was ran at 90 Volts during 100 minutes. The first well contains a 1 kb ladder. The third well contains 5 µl of Fabricio's extraction, the fifth well contains 5 µl of my extraction; I used 3 µl of dye for the last two.
  • Besides the fact that the ladder can barely be seen, none of the two plasmids can be seen. I will ask if this ethidium bromide concentration is correct.



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