Team:TorontoMaRSDiscovery/Notebook

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Home The Team The Project Parts Submitted to the Registry Modeling Notebook


Contents

April 27, 2009

  1. Received from Rosa (SPiT):
    • TM0785
      • Plasmid containing encapsulin
      • Recommend transfect into bacteria and re-sequence
      • See email note regarding sequence error
    • 0.5 microliters TMG DNA 100 microgram/microliter
      • Use 0.4 microliter for 50 microliter PCR reaction
      • This is thumotoga maritime genomic DNA for purpose of re-cloning
  2. Microcentrifuge tubes 1 and 2 placed in -20 freezer

May 15, 2009

  1. pH buffers received from VWR Mississauga
    • pH 4 buffer (red) 500 ml
    • pH 7 buffer (yellow) 500 ml
    • pH 10 buffer (blue) 500 ml
      Above are used for pH/mV Meter calibration
  2. pH/mV meter calibrated according to manual – recorded in index
  3. Ethanol solution (70%) made from 85% ethanol

May 19, 2009

  1. 2L of TE buffer made (10X TE)
    Recipe for 2L from stock solution (10X TE)
    a) 10 ml 1M Tris-HCl pH 7.5-80.0 x 2 = 20 ml
    b) 2 ml 0.5 M EDTA pH 8.0 x 2 = 4 ml
    c) 988 ml ddH20 x 2 = 1976 ml
    To make Tris-HCl, Tris base is used and adjusted to desired pH using HCl
    1 M Tris base is required of which 20 ml is required – thereby 2.4228 g Tris base (due to scale limitations, 2.42g of Tris Base was used
  2. 500 ml of 1 M Tris Base made
    mass of Tris used = 1M/1000 ml x 500 ml x 121.14 g/mol = 60.57 g
    volume of water used = 500 ml
  3. 250 ml of 0.5 M EDTA solution was made
    mass of EDTA used = 36.53 g
    observations: EDTA did not dissolve in ddH2O on heat and being stirred

May 21, 2009

  1. Retrieved autoclaved ddH20, glycerol solution
  2. Gel Electrophoresis (test run)
    • 1% gel: 0.5 g agar mixed with 50 ml of 1X TBE
    • 10X TBE diluted with 5 ml TBE + 45 ml ddH20 – did not use all 50 ml for 1 gel (gel will be too thick – make 2 instead)
    • Loading Dye: add glycerol solution to 25 mg bromophenol blue then add dH20 to 10 ml (to make 6X)
    • Running gel: match wells to black side, run at 120 mA
  3. Visualize Gel in UV
    1. Turn power on
    2. Gel in machine face up
    3. Close door securely
    4. Turn white light on
    5. Adjust zoom, contrast, focus from black dial on top of machine
    6. Turn white light off (turns on UV)
    7. Press ‘live’ toggle – acq. Should be 0.4 sec.
    8. Print if desired or save on floppy disk
    9. Turn power off
    10. Dispose of gel in proper container
    11. Close door

May 25, 2009

  1. Made overnight bacteria culture (200 ml LB: 20 microliters DB3.1)

May 26, 2009

  1. Took overnight cultures from incubator
  2. Inoculated 2 500 ml flasks with 25 ml of overnight culture (1 flask/culture)
  3. Placed 500 ml flasks into incubator at 37 degrees Celcius
  4. Grew overnights of DB3.1 from Waterloo (thanks :))

June 3, 2009

  1. Plasmid transformed = pSB1AC3 (TEST)
    • Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.

June 5, 2009

  1. Tet plates made
    Recipe for 200 ml (approx. 10 plates):
    2.2 g agar in 200 ml fresh LB
    Note: do not re-autoclave LB, it will caramelize!
    Recipe for 200 ml LB:
    a) 1 g yeast extract
    b) 2 g peptotryptone
    c) 2 g NaCl
    d) 200 ml water
  2. Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
  3. Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
  4. Swirl and poured into prepared, labeled plates
    • Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
  5. Inverted and put in 37 degree incubator to dry

June 8, 2009

  1. Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
  2. Bacterial liquid culture placed in shaker at 10:51 a.m.

June 9, 2009

  1. Digested miniprepped gel with EcoRI and SpeI
  2. Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
  3. DNA Ladder made - 6 microlitres of stock used

June 10, 2009

  1. Poured 10 Tet plates following procedure on June 5, 2009
  2. Due to problems with DNA ladder and restriction enzyme digest yesterday, procedures were followed again today with the following adjustments
    • DNA was diluted and run on lanes 1-5 of gel:
      • Lane 1 - 1X
      • Lane 2 - 1/6X
      • Lane 3 - 1/36X
      • Lane 4 - 1/10X
      • Lane 5 - 1/100X
    • Restriction enzyme digest was repeated with an incubation time of 4 h at 37 degrees Celsius and heat shock at 80 degrees Celsius was done in hot water bath
    • Results: Dilution did not work well (ladder could not be visualized). Digest appeared to work as 3 bands could be seen in last 2 lanes
    • Adjustments for tomorrow:
      • Spin down enzymes before using
      • Overnight digest

August 1, 2009

  1. Overnight encapsulin cultures 1 and 6 (randomly selected) and the negative control were miniprepped
  2. The rest of the encapsulin cultures were stocked with 20% glycerol
  3. 6 digestions were done: Enc1, Enc6, Enc -ve, BB5, BB7, and C
    • 16ul of BB5 plasmid was used
    • 500ng of plasmid were used for the others
  4. The digestions were run on a 1.3% agarose gel in TAE
    • BB5 was confirmed and all other parts were correct as well
  5. Overnight ligation of 7+Enc in the PCR machine
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