Team:UBrawijaya/Notebook

Team:UBrawijaya - 2021.igem.org

Notebook

CULTIVATION & PRESERVATION MICROBE
  • Cultivating DH5 alpha/BL21/BL21 (DE3) from the broth medium to the slant agar for storage
      • Protocol:
    1. Sterilize the ose and put it into a briefly vortexed cultivation broth
    2. Streak the ose into agar
    3. Incubate 12-18 hours 370 C in an incubator
    4. Store on the refrigerator at 40 C
  • Cultivating DH5 alpha/BL21/BL21 (DE3) slant medium into broth medium
      • Protocol:
      1. Sterile the ose third time with alcohol 70% and burnt the ose on fire (bunsen) until the ring is changed to the red
      2. Collect one colony on slant medium and transfer into 30 mL LB broth as a starter for the next step of competent cell protoco
      3. Incubated 12-18 hours 370C in incubator or shaker waterbath using 120 rpm 370C
      4. Incubated 12-18 hours 370C in incubator or shaker waterbath using 120 rpm 370C
    • Preservation recombinant DH5 alpha/BL21/BL21 (DE3)
        • Protocol:
        1. CTransfer 400 µL culture and add 200 µL glycerol 60% into the eppendorf tube sterile (final concentration 20% preservation)
        2. Gently pipette
        3. Store on deep freezer (-80oC)

    PCR
  • Resuspended Primer IDT
      • Protocol: base on IDT protocol
    1. Prior to opening, centrifuge the tube at a minimum of 3000xg to ensure that the material is at the bottom of the tube
    2. Add TE buffer/dH2O (distilled water)/NFW (nucleus free water) which amounts to …. µL that calculated from nmole of the primer (*each primer have different nmole) you able to use calculator IDT tool in https://sg.idtdna.com/calc/analyzer and choose resuspension calculator tool
    3. If you want to dilution the shock you able to calculated on the same link and choose dilution calculator
    4. Mix the TE buffer/dH2O/NFW and primer stock on a sterile microtube
    5. Serial dilution (optional)
    6. Storage -200 C

    Label A

    Forward plasmid backbone pSB1C3 & pSB1K3
    27.2 nmoles + 272 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW


    Label B

    Reverse plasmid backbone pSB1C3 & pSB1K3
    26.9 nmoles + 269 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW


    Label C

    Forward (1) backbone overhang pSB1C3
    26.5 nmoles + 265 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW


    Label D

    Reverse (1) backbone overhang pSB1C3
    19.4 nmoles + 194 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW


    Label E

    Forward (2) insert pSB1C3
    29 nmoles + 290 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW


    Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base


    Label F

    Reverse (2) insert pSB1C3
    24.2 nmoles + 242 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW
    Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base.


    Label G

    Forward (1) backbone overhang pSB1K3
    22 nmoles + 220 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW


    Label H

    Reverse (1) backbone overhang pSB1K3
    22.8 nmoles + 228 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW


    Label I

    Forward (2) insert pSB1K3
    27.8 nmoles + 278 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

    Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base.


    Label J

    Reverse (2) insert pSB1K3
    26.5 nmoles + 265 µL NFW → stock concentration 100 µM
    Stock dilution into working concentration 10 µM → add 10 µL stock 100 µM + 90 µL NFW

    Note: because in our design the insert circuit includes an overhang base on the suffix and prefix part, so the primer design haven’t overhang base.


  • Resuspende gblock insert pSB1C3
      • Protocol: base on IDT protocol
      1. Prior to opening, centrifuge the tube at a minimum of
      2. 3000xg to ensure that the material is at the bottom of the tube
      3. Add TE buffer/dH2O/NFW to reach a final concentration 10ng/µL
      4. Vortex briefly
      5. Incubate at 50oC for 20 minutes
      6. Briefly vortex and centrifuge
      7. Serial dilution (optional)
      8. Storage -20oC
    • PCR reaction
        • Protocol: base on NEB protocoll
        1. Mix 25µL volume reaction, add
          10 µL Forward (FWD) (label E) 1.25 µL
          10 µL Reverse (REV) (label F) 1.25 µL
          Template DNA 1 µL
          NEB Q5 master mix 12.5 µL
          NFW 9 µ
        2. Spin down for a second
        3. Put PCR eppendorf into PCR mechanine
        4. Setting condition PCR 30 cycles
          98oC 30s
          98oC 10s
          ? 30s → annealing temperature depends on primer
          72oC 30s
          72oC 2 mins
          4oC 5 mins

        note: if running gradient PCR annealing temperature using multi-temperature (56oC-72oC ) to get optimal annealing temperature

    CLEANUP PCR
  • DNA PCR cleanup using kit NEB [PCR & DNA Cleanup Kit (5µg)]
      • Protocol: Base on NEB protocol
    1. Dilute sample with DNA cleanup binding buffer 5 : 1 sample [dsDNA < 2 kb] for amplicon product then mix well by pipetting (do not vortex)
    2. Insert column into the collection tube and load sample onto the column
    3. Spin 13.000 rpm 1 minute, then discard flow-through
    4. Re-insert column into the collection tube and add 200 µL DNA wash buffer
    5. Spin 13.000 rpm 1 minute, then discard flow-through
    6. Repeat steps 4-5
    7. Repeat steps 4-5
    8. Add 20 µL of DNA Elution buffer to the center of the matrix and wait 1 minute
    9. Spin 13.000 rpm 1 minute, then remove the filter tube and DNA elute on the 1.5 mL microfuge tube
    10. Measure concentration and purification DNA with nanodrop

    ELECTROPHORESIS
  • Electrophoresis DNA
      • Protocol:
    1. Made 1.5 % agarose gel and add 1x TBE buffer solves then heat it around 4-5 minutes next pour into the electrophoresis plate print and wait until the gel hardens
    2. Prepare and mix components for marker well:
      Ladder 1 Kb 4 µL
      Gel red 2 µL
    3. Prepare and mix components for the sample well:
      Loading dye 2 µL
      Gel Red (DNA staining) 2 µL
      DNA (>100 ng/µL) 1-5 µL → PCR product
    4. Transfer the gel into the electrophoresis equipment and pour the 1x TBE buffer until the gel submerged
    5. Loading the marker and the DNA sample into the well
    6. Running by 50 volts for 60 minutes
    7. Visualized the gel with gel dock

    PLASMID ISOLATION
  • Plasmid isolation using NEB kit [Monarch plasmid miniprep kit]
      • Protocol: base on NEB protocol
    1. 1 mL - 5 mL culture do centrifugation for 30 seconds 13.000 rpm and get the pellet for isolation
    2. Resuspended pellet with add 200 µL plasmid resuspension buffer (B1) then pipet up and down to ensure cells are completely resuspended
    3. Add 100 µL plasmid lysis buffer (B2) and gently invert the tube around 5-6 times, then incubate 1 minute at room temperature
    4. Add 400 µL plasmid neutralization buffer (B3) and gently invert the tube until neutralized, then incubate 2 minutes at room temperature
    5. Centrifuge lysate for 2-5 minutes 13.000 rpm
    6. Transfer supernatant to the spin column and centrifuge for 1 minute 13.000 rpm, discard flow-through
    7. Re-insert column into the collection tube and add 200 µL DNA wash buffer 1 and centrifuge for 1 minute 13.000 rpm, then discard flow-through
    8. Add 400 µL plasmid wash buffer 2 and centrifuge for 1 minute 13.000 rpm
    9. Transfer column to a clean 1.5 mL microfuge tube
    10. Add 30 µL of DNA Elution buffer to the center of the matrix and wait 1 minute
    11. Spin 13.000 rpm 1 minute, then remove the filter tube and DNA elute on the 1.5 mL microfuge tube
    12. Measure concentration and purification DNA with nanodrop

    ASSEMBLY & TRANSFORMATION
  • Gibson Assembly and Transformation [NEBuilder HiFi DNA Assembly Cloning Kit]
      • Protocol: base on NEB protocol
    1. Set up following reaction on ice
      Insert DNA length = …...bp
      Vector DNA length = …...bp
      Vector DNA mass = …... ng/µL → 1 µL
      (required insert DNA mass …...ng [3:1])
    2. Calculation of how much µL for fulfilling required insert DNA mass …..ng → …..µL
    3. Add 2-3 fragment assembly with recommended DNA ratio (3:1) and mix all reactions with pipetting
      Total amount fragment X µL
      NEBuilder HiFi 10 µL
      NFW 10-X
      Total volume 20 µL
    4. Incubated the sample in a thermocycler at 50oC for 15 minutes and then storage at -20oC for a moment or around 15-30 minutes
    5. Thaw chemically competent cells on ice
    6. Add 2 µL of the chilled assembled product to the competent cells, then mix gently by pipetting up and down or by flicking the tube 4-5 times. Do not vortex
    7. Place the mixture on ice for 30 minutes. Do not mix
    8. Heat shock at 42oC for 30 seconds. Do not mix
    9. Transfer tubes to ice for 2 minutes
    10. Add 950 µL of room-temperature SOC media for DH5 alpha and LB media for BL21/BL21 (DE3) into the tube
    11. Incubate the tube at 370C for 60 minutes. Shake vigorously (250 rpm) or rotate
    12. Warm selection plate to 370C
    13. Spread 100 µL of the cells onto the selection plates. Use CHL plates for the positive control sample
    14. ncubate overnight (12-18 hours) at 370C
  • screening selection media
      • Protocol:
    1. Pipet antibiotic chloramphenicol 33 µg/mL or kanamycin 30 µg/mL (1 mL of medium add 1 µL of antibiotic)
    2. spread the mixture evenly across the plate
    3. Incubate at 37°C overnight or approximately 12-18 hours