User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/05/16

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χρόνος πέρασμα May 16th 2011

  • Again, I made an electrophoresis gel to see if I correctly extracted the plasmids pBBRMCS5 and pSB4C5 that were isolated last week.
  • I used the last 5 microliters (there is no more plasmid) and followed the same procotol as the other gels I've run. The only difference is that I set the electrophoresis chamber to 100 volts and to 70 minutes. Approximately at 6:15 p.m. I was about to stain the gel with ethidium bromide when some labmates told me the reason I couldn't clearly see something in the last gels was that I hadn't left the extractions to be digested to linearize the plasmids. The fuzzy and continuous banding observed is due to the several isoforms (concatamers) that exist in plasmidic DNA.
  • Nonetheless, this is the gel:
PBBRMCS5 pSB4C5 gel4.jpg
  • Next time, I surely will not forget to digest the extracted plasmids. I left incubating more DH5α E. coli carrying the plasmids in liquid medium overnight. 4 test tubes were used per each plasmid, with 5 ml of liquid medium. 3 test tubes (for each plasmid) were added with the respective antibiotic (5 microliters of chroramphenicol and 5 microliters of gentamicin, for pSB4C5 and pBBRMCS5, respectevely). The 4th test tubes were added with the proper bacteria without the antibiotic.