Team:Illinois/Bimolecular Fluorescence Biosensor Notebook

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Contents

1st July

  • TE Buffer Recipe
    • Uses
      • TE used to bring up oligos into solution
    • People who know how to do this
      • Joleen, Luke
    • Concentration
      • 10mM Tris
      • 1mM EDTA
      • pH 8.0
    • Method
      • For 500mL volume
        • Add: 0.1861g EDTA; 0.6057g Tris to 1 liter flask
        • Bring it up to 500mL with Deionized water (filtered in ---? container)
        • Put on "corning" mixer; get a larger stir bar from drawer and put on slow ~20 RPM rotation; no heat for 2-3 minutes until fully dissolved.
        • Standardize the pH meter (instructions on sign at prep bench)
        • Put gloves on; put pH electrode in flask but first add stir bar and put on low speed mixing
        • Bring pH to 8.0 (i.e. 8.00 +/- 0.05) by adding base (NaOH) or acid (HCl) in very small drops using a plastic pipette. (Wait for pH meter to eqiullibrate after each drop)

IMP: Put cap back on bottom of pH probe

        • Put coloured tape along side of flask and label with pH, name and iGEM
        • Cover it with heavy duty aluminium foil (just like a square inch to cover the top) and put a strip of autoclave tape along top of foil
        • Optional: out in big plastic autoclave bin
        • Bring to autoclave room; do not use the big "Beta Star" 
    Set on: 15 minutes; ~250 degrees Farenheit = ~121 degree Celcius; "liquid" run; for "operator #" just press enter; then "Run"; tubes = ~ 45 minutes; total to run since it must cool down and decompress
        • (Some extra side notes still to add)
  • TAE Buffer Recipe
  • TBE Buffer Recipe
      • 1 liter of 5x TBE Running Buffer, pH 8.13-8.23
    • Materials
      • Tris-base - 54.0g
      • Boric Acid - 27.5g
      • EDTA - 2.92g
      • DI Water - 1.0L
    • Method
      • Stirred with stirring rod for 3-5 minutes
  • Agarose Gel

2nd July

  • DNA Sequencing - "Core Sequencing " --?
    • (https://unicorm.biotec.uiuc.edu) -> "create login account" -> go to "payment manager"
    • Primer: One end primer, included in per tube, 10uM concentration of primer
    • 30-40 ng/uL? sample concentration
    • Protocols/prep online
    • ---? to view chromatograms on site
    • $5 for sequencing; $2.50 for "--? to load"
    • Purify with PAGE before bringing in

8th July

  • Agarose Gel Purification of Oligos
    • Made 4% Agarose gel with 8g Agarose, 40mL TBE, 160mL H2O
    • Made 2% with 4g Agarose
    • Microwave for 1:35 for optional warming
    • Ran total of 2 hours on 80 Volts -> --? (decent?)
  • TAZ side project
    • Adam Z streaked 4 plates with High Elu, Amp m100, Taz Ecoli
    • Check for growth tomorrow
  • Gel Extraction Protocol
    • Use microscope slide to cut gel out
    • Visualize on Bio unit with plastic shield from drawer below unit attached to slide out unit
    • Amount of gel collected(into 1.5mL tubes)
    • Oligo #2: 0.0671g -> 402.6g 
  Oligo #3: 0.062g -> 362.0g
  Oligo #4: 0.1062g -> 637.2g
  Oligo #5: 0.0747g -> 448.2g
  Oligo #6: 0.0585g -> 351.0g
  Oligo #7: 0.0601g -> 360.6g
  Oligo #8: 0.0440g -> 264.0g
  Oligo #9: 0.0482g -> 289.2g

9th July

    • Cells were found in small colonies -> decided to let them grow for 24 more hours
    • Plated 4 more E.coli strains with Xgel (spread)
    • Cultured 4 vials of E.coli with X gel
    • Check back in 36 hours




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