User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/05/12

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χρόνος πέρασμα May 12th 2011

  • The plasmids I isolated yesterday were not left to incubate with the TE RNAse for too long (actually, just for ~10 minutes) as some people have told me it did not make any difference. I added 20 µl of TE RNAse.
  • Today I made an electrophoresis gel to see if the plasmids had been correctly isolated. A flask that already contained the gel was just heated during 3 minutes with a microwave potency of 90 to melt the gel. I put 5 µl of the plasmid solution along with 3 µl of the dye. I used two of the wells to put the ladder, even though I knew it wouldn't serve to see the plasmid size as the plasmids remained circular and wouldn't behave the same as if they were digested (linearized); the ladder was put as a control to see if the ethidium bromide did marked DNA molecules. The gel was ran ~80 minutes at 100 volts.
  • I accidentally tore the gel many times. Fortunately, I still was able to see it clearly.
  • The gel had 15 wells. #1 and #15 contained the ladder. #2, #7, #9, #13 and #14 were empty. #3-#6 had the plasmid extraction pBBRMCS5. #8 had only the dye without DNA. #10-#12 had the plasmid extraction pSB4C5. As it can be seen, the negative control (#8) and the positive controls (#1 and #15) were successful. The problem is that the plasmids didn't appear to be completely isolated, as many things were dyed with the ethidium bromide. Fibo hypothesized the inespecific ethidium bromide staining was due to the short period of TE RNAse incubation time. He left 15 µl left extraction tubes to incubate during ~half an hour.
  • pSB4C5 is a BioBrick standard vector with low copy pSC101 replication origin (copy number ~5) and chloramphenicol antibiotic resistance marker.
  • pBBRMCS5 is a relatively small broad-host-range vector (>5.3 kb), possess an extended multiple cloning site, has a gentamicin resistance marker and is reported to reside inside bacteria with a copy number of ~30.

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