Team:NRP-UEA-Norwich/Notebook Protocols

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NRP UEA Norwich iGEM 2014

Lab Protocols

As students we have learnt many new skills and techniques whilst completing our iGEM project. We have documented the protocols we have used to successfully create a biosensor for plant pathogen interactions using Golden Gate Cloning.

GoldenGate Digestion-Ligation reaction (Level 1)

Aim: to form a level 1 construct (full transcriptional unit) from level 0 modules in a in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.

  • Add relevant DNA to a PCR tube at a 2:1 ratio of insert to acceptor (dilutions may be required depending on starting DNA concentration).
  • Add enzymes and reaction buffers according to the template shown below.
  • Make up the reaction mix to 15 µL using distilled water.
  • Reaction component Volume(µL)
    Level 1 acceptor Ratio 2:1 (Insert: acceptor)
    Insert 1 Ratio 2:1 (Insert: acceptor)
    Insert 2 Ratio 2:1 (Insert: acceptor)
    Insert 3 Ratio 2:1 (Insert: acceptor)
    T4 reaction buffer 1.5
    BSA buffer 1.5
    BsaI 0.5
    T4 DNA Ligase 0.5


  • Cycle the reaction mix in a PCR machine according to the program given below.

  • Step Temperature (°C) Time (minutes: seconds)
    1 37 0:20
    2* 37 3:00
    3* 16 4:00
    4 50 5:00
    5 80 5:00
    6 16

    *Steps 2-3 cycled x26

    GoldenGate Digestion-Ligation reaction (Level 2)

    PROTOCOL HERE

    Making LB Agar

    PROTOCOL HERE

    Making LB broth

    PROTOCOL HERE

    E. coli Calcium Chloride Heat Shock Transformation

    Aim: to get DNA expression in E. coli.

    • Remove chemically competent E. coli cells from the -80oC freezer and thaw on ice.
    • Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.
    • Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.
    • Preheat water bath to 42oC.
    • Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).
    • Transfer back onto ice for 5 mins.
    • Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.
    • Spread plate 100 µL onto plates containing the relevant antibiotics and incubate over night at 37oC.

    E. coli Electroporation Transformation

    PROTOCOL HERE

    Making electrocompetent Agrobacterium tumefaciens

    PROTOCOL HERE

    Agrobacterium tumefaciens Electroporation Transformation

    PROTOCOL HERE

    Blue- White Selection

    PROTOCOL HERE

    Colony PCR

    Aim: to determine the size (bp) of the DNA expressed by the colony picked

    This helps us to check that the construct is what we expected.

    DNA mini-prep

    Aim

    The following protocols were used with QIAGEN QIAprep miniprep kits.
    • Create an overnight culture- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with shaking.
    • Centrifuge 1-5ml of overnight culture at >8000 for 3 minutes in an Eppendorf tube to form a bacterial pellet; discard the supernatant.
    • Re-suspend bacterial pellet in 250µl of P1 Buffer (kept at <5oC).
    • Add 250µl of P2 Buffer and invert 4-6 times to mix thoroughly. This reaction is left for no longer than 5 minutes before completing the next step.
    • Add 350µl of N3 Buffer and invert 4-6 times to mix thoroughly.
    • Centrifuge for 10 minutes at 13,000rpm
    • Decant supernatant into a spin column, centrifuge for 30-60 seconds, and discard the flow-through.
    • Add 750µl of PE Buffer to the spin column and centrifuge for 30-60 seconds to wash the DNA. Discard the flow-through and centrifuge for a further 1 minute to remove any remaining buffer.
    • Remove the top section of the spin column and place in a clean 1.5ml Eppendorf tube. Add 50µl of EB Buffer to the column; let it stand for 1 minute before centrifuging for a further 1 minute to elute the DNA.

    Preparation for sequencing

    Aim: to sequence our DNA in order to check content of our constructs

    • Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes.
    • Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)
    • Send off both 10µl samples for sequencing.

    Agrobacterium tumefaciens Infiltration

    PROTOCOL HERE

    Infiltration Analysis

    PROTOCOL HERE

    Making GoldenGate compatible PSB1C3 'Flipper'

    PROTOCOL HERE

    Antibiotic Selection

    PROTOCOL HERE
    A big thank you to our sponsors