Difference between revisions of "Team:UrbanTundra Edmonton/Notebook"

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<h1> Our Lab Notebook </h1>
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<p> View the our progress and latest developments, all on a daily basis. </p>
  
 
     <h3>July 20th, 2016</h3>
 
     <h3>July 20th, 2016</h3>

Revision as of 17:27, 17 October 2016

URBAN TUNDRA ▤

Our Lab Notebook

View the our progress and latest developments, all on a daily basis.

July 20th, 2016

  1. 4 microlitres of milliq water with 1 microlitre of plasmid
  2. 5 microlitres of no chlorite dismutase
  3. 1 microlitre of cutsmart buffer (specifically 1.1111 microlitre)
      4. A. 10 x cutsmart buffer into tubes makes 1x [ ]
  4. When adding BsaI, only touch the surface of the BsaI to the pipette tip. The reason: has glycerol which is sticky. Take 0.5 microlitre of BsaI.
  5. Mix sample very gently then put into a 37 degrees celsius incubator for 1 hour.
  6. Heat kill at 65 degrees celsius for 20 minutes. This kills the BsaI to prevent star activity.
  7. Pulse down tubes then add 8 microlitre of milliq water.
  8. Add 2 microlitres of T4DNA ligase buffer.
  9. Add 0.5 microlitre of ligase then mix gently and incubate at room temperature for 30 minutes or longer.


July 21st, 2016

  1. Did some plating.
  2. Plated three agar types:
      3. A. 4 plates of no antibiotic
      B. 10 plates of kanamycin (CM)
      C. 10 plates of ampicillin (Amp)
  3. Purpose: try to find the antibiotic marker on bacteria.
  4. Observational expectations: If it’s purple, then there is no Cld gene sequence. If white then has CLd gene sequence.
  5. Lives or dies on certain plates. Alive on ampicillin, then it has the marker. Dies it has the CM marker.
  6. Added 20 microlitres in each plate
      7. A. A total of 7 plates
      B. DNA minus has none, CM, and AMP
      C. Positive control has CM and AMP
      D. Cld +/- has CM and AMP

Competent cell transformation:

  1. 1 microlitre of positive control added to competent cells (positive control: CPB we used yesterday before cutting and gluing)
  2. 10 microlitre of cloned plasmid added to competent cells (Cloned plasmid is what we did yesterday).
  3. Positive control, negative control, and cloned plasmid were chilled for 30 minutes, heat shocked for 90 seconds, chilled on ice for 5 minutes, and finally incubated at 37 degrees celsius with 1 ml of LB broth.


July 22nd, 2016

  1. It was observed that there were little purple colonies, however the colors will develope more over a longer period.
  2. Results from yesterday:
      3. A. No Ab plates: very thick growth
      B. Amp plates: no growth: conclusion: no amp marker
      C. CM: growth
  3. Results chart:
    4. + control DNA - Cld + Cld -
    No Antibiotic Yes, very thick
    Amp No growth No growth No growth No growth
    CM Growth (purple colonies) No growth Possibly purple and white* Possible purple and white*

* Purple colour will develop more over larger period of time (perhaps over the weekend)



July 23rd, 2016

Today was spent trying to get sponsors.



July 25th, 2016

Lab work: Today we created overnight cultures through inoculation for miniprep tomorrow

  1. Pipette 5 ml of LB broth into 6 tubes
  2. Add CM for antibiotic in the Broth
      3. A. Had to do the above procedure again
  3. Have 60 ml of LB broth
      4. A. One tube gets 50 ml → 50 microlitres of CM.
  4. Put 5 ml into culture tube
  5. Inoculate bacteria grown from last lab day.

    6. -CM allows only bacterias with CM markers to exist, thus allowing only organisms with the designated sequence to survive and live.

After that, more fundraising work was done.



July 26th, 2016

  1. Yesterday’s labwork had to be redone since the inoculation wasn’t done correctly.
  2. Reason: pipette tips used were too short and because the top of the tips were contaminated, the whole culture became contaminated.
  3. In our redone experiment, we used inoculation stick instead (‘just to play it safe’ Mike reasoned)


July 27th, 2016

    Miniprep day

    Cld+ cultures from yesterday were purple- that’s not good (we want white ones) so we re-inoculated Cld+ for another miniprep tomorrow

    Cld- cultures from yesterday were good so we are able to perform miniprep today with the Cld-

  1. Microcentrifuge 1 ml of culture (do this three times to allow a large amount of cells to build up)
      2. A. These cells should be orange-reddish in colour. The reason for this is due to the production of a ‘heme’ group- that is a protein that binds with Fe.
  2. Addition of 250 microlitres of Buffer-PI allows resuspension of bacteria.
      3. A. Buffer PI was spilled on the table, but another bottle was brought down.
      B. To thoroughly mix it, vortex the microcentrifuge column.
  3. Add 250 microlitres of Buffer P2. Mix by inverting it 4-6 times.
      4. A. Don’t let this sit for more than 5 minutes.
  4. Add 350 microlitres of Buffer N3 and mix by inverting 4-6 times.
      5. A. A whitish precipitate should have formed.
  5. Microcentrifuge for 10 minutes at >10 000 rpm.
      6. A. Supernatant is what we want. The solid is the garbage.
      B. Once taken out, supernatant will be pipetted out into a separate tube (not a spin column). This allows unison of the supernatant and consistency
      C. *Note* Eppendorf tubes are microcentrifuge tubes
  6. Pipette out supernatant from new microcentrifuge into labeled spin columns
      7. A. Centrifuge it for 30-60 seconds
      B. Let it sit for a bit
  7. Add 0.75 ml of Buffer PE and centrifuge for 30-60s.
      8. A. Discard flow-through and centrifuge one more time for 1 minute.
      B. Residual wash buffer (remove it)
  8. Add 100 microlitres of Buffer EB after transferring spin column over a new microcentrifuge tube.
      9. A. Stand for one minute
      B. Spin for one minute
      C. *Technique* Using finger to balance it and place it just above the filtre- Don’t touch the filter.
  9. Now, we will test concentration and purity through the use of a spectrophotometer.
  10. Results:
    11. Vial Number 260/280 Concentration in nanograms/microlitre
    Read 1 1 1.77 88.4
    Read 2 1 1.80 85.0
    Read 1 2 1.81 84.4
    Read 2 2 1.80 85.0
    Read 1 3 1.79 87.0
    Read 2 3 1.81 78.6
    Read 1 4 1.79 83.6
    Read 2 4 1.79 83.1
    Read 1 5 1.79 77.7
    Read 2 5 1.77 81.2
    Read 1 6 1.81 76.7
    Read 2 6 1.79 77.2


July 28th 2016

    Miniprep (again) today

  1. Cld + cultures are ready (not purple this time)
      2. A. Perform miniprep on them
      B. We went through the exact procedure as the previous day

Miniprep

  1. Observations: Like yesterday, the pellet that formed after the microcentrifugation is reddish-orange
      2. A. Vial 4 (after vortex with Buffer PI) appears to be purplish in colour rather than pink like the rest.
      B. Pink: “Strawberry Milkshake”
      C. Purple: “Glove coloured purple with a slight tint of pink”
  2. Second centrifugation (with PE) of number 4: the waste was blue and this might be due to the blue dye of the sharpie we used to differentiate vial 4’s content from the rest.
      3. A. At first, it was believed this was due to the initial purple colour, but upon closer inspection, it was realized some blue streaked marks suggested that enthanol cleaned away the blue sharpie dye.
  3. Results:
    4. Vial Number 260/280 Concentration in nanograms/microlitre
    Read 1 1 1.78 86.7
    Read 2 1 1.83 85.4
    Read 1 2 1.79 98.5
    Read 2 2 1.79 98.6
    Read 1 3 1.83 81.9
    Read 2 3 N/A N/A
    Read 3 3 1.82 82.9
    Read 1 4 1.82 97.1
    Read 2 4 1.80 97.3
    Read 1 5 1.82 92.7
    Read 2 5 1.82 93.9
    Read 1 6 1.82 95.4
    Read 2 6 1.82 95.4
      A. *N/A is due to pipette contamination. The 260/280 value here was 1.64 and the concentration reading was 93.8 nanograms/microlitre
      B. Cld+ may have higher concentrations due to histidines present in the Cld+ sequence.
  4. Next, we created a recipe for mixmaster:
      5. A. It was calculated through the formula C1V1=C2V2 that the it was required 26.666… or 27 microlitres of cutsmart. 171 microlitres of milliculewater and 2 microlitres of BsaI.
  5. Gel Electrophoresis
      6. A. To check the plasmid backbone and the Cld +/- insert.
      B. Separate (and no gluing)
  6. Create master mix by adding 27 microlitres of cutsmart, 171 microlitres of millicule water and 2 microlitres of BsaI. *remember that enzymes are always kept on ice!*
  7. Transfer fifteen microlitres of master mix into each digestion tube (a total of 13 tubes)
      8. A. Each tube is labeled with a digit from 1 to 6 and whether it was Cld - or +.
  8. Add 5 microlitres of DNA to each tube.
      9. A. *note: cutsmart buffer is used to provide optimal environment for cut enzyme.
      B. Cut DNA to compare relative sizes: we will see:
      a. Cld +/- (1 Kb)
      b. Colour gene (0.7 Kb)
      c. Whole plasmid backbone (4 Kb)
    ~Lunch break~
  9. Gel electrophoresis
      10. A. Make gel: 1% agarose in TAE into well
      a. *1% is 1g/100ml
      B. Add loading dye into tubes
      C. Add 5 microlitres of sample into wells
      D. Same procedure as practice procedures found online
  10. Pcitures of results are taken....


August 2nd, 2016

  1. We split the team up into non-science and science groups...


August 3rd, 2016

    • Science based
      • Perchlorate team initiates
      • Biobrick team initiates
      • Process and quant bio are to design the experiments and must research up on it. (This requires some time)
    • Non science based
      • Fundraising work was done
    • Biobrick team: tomorrow, they will perform miniprepe after inoculating bacteria replicated today.
      • Today, chemical transformation was performed (refer to 8.5) to create a lot of plasmids (there is not enough)
          • *When referring, note that ligase was not added
        • Plasmid that were used were those given from igem (required in order to win gold).
        • Today, inoculation of grown bacteria will be performed to create cultures for miniprep tomorrow.
      • Chloramphenicol: antibiotic will be added to the inoculated cultures.
        • The given plasmids have a chloramphenicol resistance marker.
      • All in all: the purpose of today for the biobrick team: Create more plasmids because there isn’t enough.
        • Then. create cultures for miniprep.
    • Procedure of the Biobrick team:
    1. Take out chemically competent e.coli from freezer and put in ice. 50 microlitres of e.coli into microcentrifuge tube.
    2. DNA are added to cells. Stir with pipette tip, but do not pipette up or down.
    3. Incubate on ice for 30 minutes.
    4. Heat shock at 42 degrees celsius for 30 seconds.
    5. Incubate for 5 minutes on ice, then add 1 ml of LB.
    6. Incubate for 1 hr at 37 degrees celsius at 250 rpm. (5ml of LB and 5 microlitres of Antibiotic)
    7. 2 culture tubes: 5 ml of LB, 15 microlitres of antibiotic, and 100 microlitres of plasmid.
    8. Incubate for whole night.


August 4th, 2016

  • Inoculation from yesterday failed
  • Therefore, we must do it again
    • Two tubes: one of each has: 10 microlitres of plasmid and 5 microlitres of antibiotic
  1. 10 microlitres of plasmid into 1 microcentrifuge tube
  2. Incubate on ice for 30 minutes
  3. Heat shock at 42 degrees celsius for 90 seconds
  4. Incubate on ice for 5 minutes
  5. Add 1 ml of LB broth into tube and incubate at 37 degrees celsius for 1 hour.
  6. Add 5 ml of LB broth into 2 culture tubes
  7. Add 5 microlitres of antibiotic into each culture tubes
  8. Transfer 200 microlitres of plasmid and e.coli into each culture tubes (Plasmid is called pSBIC3)
  9. Shake overnight at 37 degrees celsius.