Team:UT Dallas/NotebookWeek4

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biz solution

Week 4

August 1-

  • Miniprep
  • Observations: i2.11.6 grew, i2.10.7 no growth
  • Making glycerol stock
  • Dissolving and diluting primers (10 pmol/ µL)
  • PCR and gel electrophoresis and purification
  • At this stage we have both the FOFr and CheZ* with XbaI and SteI restriction sites. Our next goal will be to insert each part into a vector backbone so they will be ready for addition of the ToxR and Ctx genes. Once these two fusion products are made, we hope to transform and test whether local motion can be inhibited with CheZ and the receptor combo
  • August 2-

  • Gel electrophoresis and purification
  • Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify
  • Ligation at 16 degrees Celsius overnight
  • We added both CheZ* and FGFR each into its own vector and if the ligation just performed works we will have 2 new biobricks. The phosphorylation was done to prevent ligase from reannealing the X and S site on the vector. This means only the phosphate on the insert can be used to connect the 2 DNA sequences.
  • August 3-

  • Transformation of FOFR and CheZ*, DH5α, 50 µL 2 µL DNA at 37 degrees Celsius
  • August 4-

  • Incubation in 3 mL and chlora at 37 degrees Celsius and 220 rpm
  • Plating CheZ (K283006) and ToxR (J07009)
  • Dephosphorylation for 1 hour at 37 degrees Celsius, PCR purify
  • August 5-

  • Making glycerol stock
  • Miniprep
  • Incubation in 3 mL LB carb at 37 degrees Celsius and 220 rpm overnight
  • Test digestion ( 1 hour and 30 minutes)- about 200 ng of DNA
  • Gel electrophoresis
  • Observations: i2.15.16 looks positive, the rest are negative
  • August 6

  • Glycerol stock and miniprep
  • Image Gallery

    Notebook

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