Team:CSU Fort Collins/NoteBook/June

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CSU iGEM



6.4.16 We picked E. Coli “Stellar” cells from an LB/amp plate. The cells had been transformed with pLC71. We put the cells into test tubes with 5 mL of LB and 5 µL of ampicillin and then put those in the shaker at 37℃ overnight. Protocol for picking cells from plate to test tube:

  • Materials
  • Autoclaved test tubes
  • 100mL LB
  • 100mg/mL ampicillin
  • Test tube rack
  • Bunsen Burner
  • P10 or P20 pipette
  • Latex gloves
    • Procedure
    • Place test tubes into the rack, label, date, initial.
    • Light bunsen burner for flame umbrella.
    • In each test tube: add 5mL of LB first, then, add 5 µL of AMP. Be sure that your pipette tip is submerged in the LB when adding the AMP.
    • Remove lid from LB/AMP plate. Use a pipette tip, gently pick a colony.
    • Eject pipet tip into test tube.
    • Flame the opening of the tube and recap.
    • Incubate tubes in 37℃ for approx. 16 hours.

    6.5.16 We performed plasmid purification according to steps outlined in the S.O.P. in Tom’s lab. After purification we had ≈120 µL of plasmid solution. Fluorimetry determined the concentration of DNA was 317 ng/µL. Plasmid stored in iGEM box in fourth floor “VWR” fridge. Now it’s in the 3rd floor fridge (igem limonene box) 8.30.16 Mini-Prep Protocol (Zymo Research): Materials

    • P1 buffer
    • P2 buffer
    • P3 buffer (refrigerated)
    • (3) 1.7mL eppes.
    Procedure
    • Pour test tube contents (no more than 5mL) into (3) 1.7mL eppes.
    • Centrifuge the eppes for 3 minutes at max speed.
    • Discard supernatant, pour carefully into waste.
    • Centrifuge 1 minute.
    • Pipette and discard supernatant.
    • Add 200µL P1 buffer to 1st eppe.
    • Pipette up and down until the pellet is dissolved.
    • Pipette the buffer w/ dissolved pellet into 2nd eppe.
    • Pipette up and down until the pellet is dissolved.
    • Pipette the buffer w/ dissolved pellets into 3rd eppe.
    • Pipette up and down until the pellet is dissolved.
    • Eppe’s 1 & 2 should be empty. Discard.
    • Add 200µL P2 by shooting in.
    • Add to all of your tubes quickly.
    • Snap all closed.
    • Inversion mix.
    • Leave tubes with only P1 & P2 for no more than 5 minutes or DNA will be damaged. Be ready to add P3.
    • Add 400µL P3 (refrigerated)
    • Inversion mix until no pink remains.
    • Let sit for 1-2 minutes
    • Centrifuge full speed for 10 minutes.
    • Setup plasmid filter columns over large Zymo collection tubes.
    • Pipette supernatant into filter column.
    • Discard pellet
    • Spin column 1 minute.
    • Discard flowthrough
    • Add 200µL Endo Wash Buffer to filter
    • Spin 1 minute
    • Discard flowthrough
    • Add 400µL Plasmid DNA Wash Buffer
    • Spin 1 minute
    • Discard flowthrough
    • Spin 2 more minutes to dry (very important).
    • Move filter to labeled 1.7 mL eppes
    • Label
    • Add 16µL 10mM Tris HCl pH 8.0 to filter column
    • Let sit 2 minutes
    • Spin 1 minute
    • The flowthrough contains the DNA. Don’t throw it out.
    • (Repeat) Add 16µL 10mM Tris HCl pH 8.0 to filter column
    • Let sit 2 minutes
    • Spin 1 minute
    • The flowthrough contains the DNA. Don’t throw it out.
    • Throw away filter. Keep flowthrough in labeled eppe.

    6.6.16 60 µL of plasmid were removed from our stock and digested with SAL1 and CutSmart buffer.

    6.7.16 We ran a gel purification of our cut plasmid, stained and imaged it on the third floor. File name: limonene plasmid 1 2016-06-07 George cut our plasmid out of the gel (the largest band on the gel). Our cut gel weighed 0.48g We then used a kit to dissolve the gel and purify the plasmid. Fluorimetry showed the concentration to be 220 ng/µL

    6.8.16 first try at Infusion cloning. 35 µL Stellar E.coli and 2.5 µL of post infusion pLC71 Cells plated and left to incubate around noon. gene block: 100ng (μL/25ng) = 4μL pLC71: 100 ng (μL/220ng) = 0.45μL In-Fusion pre-mix: 2μL Distilled water: 3.55μL 50°C for 15 minutes

    6.9.16 Took transformed cells out of 4th floor incubator at 11:00 AM. No growth on either plate. Re-transforming with the same infusion mix that we made yesterday Stellar cells - 1501846A Cell & DNA mixed at 12:08 The cells were kept on an ice/water miss the whole time. The tubes where cells & DNA were mixed were also on ice before the mix. The infusion mix was stored in the eppe block for about 5 minutes. LB/amp plates by RZ 5/20/16 Heat shock at 12:34 at 44 celsius for 45s then directly back on ice. Plated at 12:40 Incubate 4th floor 49°C at 12:47

    6.10.16 Took stellar cells transformed w/ infusion mix out of the incubator at 11:40 No growth, only bubbles. Second try at infusion mix Everything on ice for 10 minutes Diluted plasmid w/ distilled water from sink.

    • 0.9uL 220ng/uL + 1.1uL dH2O
    Transformation:
    • E. Coli was pulled out of the freezer at 1:50.
    • 1:55, infusion mix was put in heat block w/ water at 50°C
    • Infusion mix was then put on ice and then mixed with E. Coli 2 minutes later.
    • We also transformed the same cells with a control of cut pLC71
    • Incubate all 3 tubes on ice for 30 minutes
    • Heat shock at 43°C for 45s
    • Plated after a 5 minutes back on ice (2:45)
    • LB/amp plates - AS 5/26/16
    • Incubated on 4th floor at 37°C overnight

    6.11.16 No growth on any plates except for 2 colonies on the control (disgested pLC71)

    6.21.16 Resuspending primers Gloves Uncap LS p1, p1000 to add 292 μL distilled autoclaved H2O from Hallies lab. A little bit of water would fall out of the pipet tip. Tip was not full. Still, only a very small amount missing. Recap.