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Revision as of 14:35, 15 November 2012 by Valerie Wisco (talk | contribs) (Cloning E.coli with the OmcF gene)
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Cloning E.coli with the OmcF gene

  • On Tuesday, we worked on the digestion of our plasmid DNA with the restriction enzymes SpecI and PstI and 10x Digestion Buffer. * We then purified the sample with the PCR purification kit to rid the sample of any additional parts.
  • To check the approximate quantity of our samples, we ran a gel along with a Low Range Ladder to judge the amounts of each sample. After this we determine the appropriate concentrations to use during ligation to help aid that process.
  • They were loaded into the well as follows:
  1. 100bp ladder
  2. 2x plasmid DNA
  3. 2x plasmid DNA
  4. 4 microL Low Range Ladder
  5. 2 microL Low Range Ladder
  6. 450bp 2x DNA
  7. 450bp 1x DNA
  8. 350bp 2x DNA
  9. 350bp 1x DNA
  10. 100bp Ladder
  • We then ligated the samples with concentrations of 4:4, 0.5:7.5, and 7.5:0.5 micro liters of plasmid DNA to our insert DNA (OmcF DNA). We also did 2 controls for each 450 and 350 DNA sample. One with no plasmid DNA and one with no insert DNA
  • On Thursday, we continued to work on the transformation, by actually combining our ligated samples with 40 micro liters of E.coli cells, icing, heat shocking, then icing the samples. We then added SOC and shook the samples for an hour.
  • We then plated our samples on solid media plates.