Team:Cardiff Wales/Protocols

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Protocols

This is a protocol for a level 0 digest/ligation reaction. Originally this was not run on a cycle as we were using BsmBI instead of Esp3I, which has an optimum temperature of 55 degrees Celsius, causing extreme disruption (but not complete denaturation) to T4 DNA ligase.

  • Set up a reaction mixture containing:
    - 1.5μl 50ng acceptor plasmid (PUDP2)
    - 3μl insert DNA
    - 1μl T4 ligase
    - 2.5μl T4 ligase buffer
    - 1μl Esp3I
    - 2μl Cutsmart buffer
  • Place reaction mixtures into a thermocycler and set up a 30X cycle of 37°C for 10 minutes then 16°C for 10 minutes

This is a protocol for a level 1 digest/ligation reaction.

  • Set up a reaction mixture containing:
    - 100ng acceptor plasmid (PGB-A2)
    - Insert DNA in a 3:1 ratio to the acceptor plasmid
    - 1μl NEB Golden gate assembly mix
    - 1μl NEB Golden gate buffer
    - Water to make the reaction up to 20μl
  • Place the reactions into a thermocycler and set up a 30X cycle of 37°C for 1 minute then 16°C for 1 minute. With a final 5 minutes at 55°C after the 30 cycles

This is a protocol for transformation of E. coli

  • Thaw 50μl aliquots of competent E. coli (DH10α) on ice
  • Add 4μl DNA to the cells
  • Leave on ice for 10 minutes
  • Place cells in a 42°C water bath for 1 minute
  • Place cells back on ice for 5 minutes
  • Add 1ml of LB broth to the reaction mixtures
  • Incubate at 37°C for 1 hour in a shaking incubator
  • Spin the cells down in a centrifuge at 13K for 1 minute
  • Remove 800μl of supernatant, then resuspend the cell pellet in the remaining 200μl of supernatant
  • Plate out the 200μl of resuspended cells and grow overnight at 37°C

This is a protocol for Agrobacterium tumefaciens transformation

  • Thaw 50μl aliquots of agrobacteria on ice
  • Add 5μl DNA to the bacteria
  • Leave on ice for 5 minutes
  • Add the reaction mixtures to liquid nitrogen for 5 minutes
  • Incubate the reaction mixtures at 37°C for 5 minutes
  • Add 1ml of LB broth to the reactions
  • Incubate at 28°C in a shaking incubator for 2 hours
  • Spin the cells down in a centrifuge at 13K for 1 minute
  • Remove 800μl of supernatant, then resuspend the cell pellet in the remaining 200μl of supernatant
  • Plate out the 200μl of resuspended cells on agar plates containing Kanamycin and Rifamycin
  • Seal the plates with parafilm and incubate at 28°C for 2 days

This is a protocol for the production of competent E. coli

    Day 1:
  • Inoculate the seed culture in 8ml of LB broth
  • Grow overnight at 37°C in the shaking incubator
    • Day 2:
  • Dilute the overnight culture 1:100 in fresh LB (800ml)
  • Grow until A650= 0.2-0.4
  • Chill the culture in an ice bath for 10 minutes
  • Harvest the cells in a centrifuge at 5K and 4°C for 10 minutes
  • Resuspend in ¼ culture volume (200ml) of 100mM MgCl2, leave on ice for 5 minutes
  • Centrifuge the culture at 4K and 4°C for 10 minutes
  • Resuspend in ¼ culture volume (50ml) of 100mM CaCl2, leave on ice for 20 minutes
  • Centrifuge the culture at 4K and 4°C for 10 minutes
  • Resuspend in 2.5ml of 100mM CaCl2, 15% glycerol
  • Make 50μl aliquots into prechilled eppie tubes, freeze in liquid nitrogen, store at -80°C

This is a protocol for the transformation of Nicotiana benthamiana

  • Spin the Agrobacteria cultures in the centrifuge at 4K for 20 minutes
  • Remove the supernatant and resuspend the pellet in induction buffer (10mM Mes, pH 5.6, 10mM MgCl2 and 150μM acetosyringone) to an OD600 of 0.5
  • Incubate for 2 hours at room temperature
  • Hand infiltrate the Agrobacteria into N. benthamiana leaves using a needle-less 1ml syringe
  • Heat shock at 37 degrees Celsius for 30minutes at 2DPI
  • Test plants of leaf discs at 4DPI

This is a protocol for making liquid broth

  • Fill a container with 400ml distilled water
  • Add 25 g/L of LB broth to the distilled water
  • Swirl/shake the bottle so that the powder has nearly dissolved
  • Autoclave the container, allow to cool, and store in fridge at 4 °C until required. Add antibiotic if needed

This is a protocol for the production of agar plates

  • Fill a container with 400 ml distilled water
  • Add 25 g/L of LB broth and 10 g/L Agar to the container
  • Swirl the container so that both the LB broth and Agar are nearly dissolved and autoclave
  • Add 1/1000th volume antibiotic (Amp, Chl, Kan etc.) to the autoclaved container along with 1/1000th volume of X-Gal or IPTG if desired
  • Distributed finalised mixture equally between 16 petri dishes, making sure all the plates are completely covered and leave to set
  • Once set, store the plates at 4 °C until required

This is a protocol for plasmid DNA minipreps using MonarchR Plasmid Miniprep Kits by NEB. For this, we followed the protocol found here.

This is a protocol for our colony PCRs to detect the presence of a desired insert

  • Pick colony of interest into 6μl ddH2O and put at 94 degrees for 5 minutes
  • Mix with 10µl PCR Super Master Mix/Taq Buffer, and 2µl of each forward and reverse primers
  • Run in thermocycler for desired cycle (we used 94 degrees Celsius for 5 minutes, then 35 cycles of 94 degrees Celsius for 30 seconds, 56 degrees Celsius (variable temperature for primers) for 30 seconds, then 72 degrees Celsius for variable time, approximately 30 seconds per Mb of DNA to be amplified), then with 72 degrees Celsius for 5 minutes, with a final hold at 16 degrees Celsius indefinitely, until ready to test

This is a protocol for agarose gel electrophoresis

  • Produce 1-1.5% Agarose gel depending on the band size you are trying to amplify, by adding agarose and microwaving for 2 and a half minutes
  • Allow solution to cool to a warm temperature and add 5μl of safeview (for a 100ml gel)
  • Pour into mould with comb and allow to set
  • Pipette 10μl of PCR mix into each well
  • Run for around 40 minutes with settings of 400mA and 100Volts

This is a protocol for the GUS staining assay

  • Cut leaf discs and place in test chamber
  • Soak for 10-15 mins in 100% acetone at room temp
  • Wash x2 with NaPO4 buffer
  • Immerse tissue is GUS staining buffer, made of:
    -50-100mm K-phosphate buffer pH7.0
    -0.05% triton X-100
    -0.25mm K3FE(CN)6
    -0.25 mM K4FE(CN)6
    -1mg/ml X-gluc

  • Vacuum infiltrate for 10minutes
  • Incubate at 37 degrees Celsius overnight or over weekend
  • Destain with 100% ethanol, changing regularly, until the leaves appear white with no chlorophyll

This is a protocol for the detection of mCherry or eGFP

  • Take whole leaf samples and place into the Photon Imager
  • Measure fluorescence of eGFP at 510nm, and mCherry at around 610nm

This is a protocol used for the detection of our siRNAs

    RNA Extraction Protocol – GenElute RNA Purfication Kit (Sigma-Aldrich) 1 – Preparation of Lysate from the Plant
  • Take circular cut outs around 1cm in diameter from leaf samples and put into 1.5mL Eppendorf
  • Freeze in liquid nitrogen (then can be stored immediately at -80°C if required)
  • Grind frozen tissue down in tube using pestle cleaned in ethanol and cooled in liquid nitrogen until tissue is a fine powder. Take care not to let the samples thaw
  • Add 600μl of Buffer RL to column and continue to grind tissue until completely homogenised
  • Pipette lysate out into a new RNase free microcentrifuge tube
  • Spin lysate for two minutes at 13,000rpm to pellet tissue debris
  • Pipette 600μl of supernatant out and transfer into a new microcentrifuge tube. To this add 600μl of 70% ethanol (100μl of 70% ethanol to 100μl of lysate). Vortex to mix

    • 2 – Binding RNA to Column
  • Insert columns into collection tubes and add 600μl of lysate/ethanol mixture directly into column
  • Centrifuge for 1 minute at 6000rpm making sure that all lysate has passed through into collection tube. (If lysate has not completely passed through spin again at 13,000rpm for 1 minute)
  • Discard the flow through and return column to collection tube

    • 3 – On Column DNA Removal (using Promega DNAse I)
  • Make up DNAse I to working volume:
    -2 μl DNAse I
    -5 μl 10x Buffer
    -43μl autoclaved H2O
    -Total = 50μl per column
  • Add 400μl of Wash Solution A to column and centrifuge for 2 minutes. Discard flow through
  • Pipette 50μl of DNAse I and centrifuge for 1 minute at 13,000rpm
  • Pipette flow through back into the column and leave to incubate for 45 minutes at room temperature
    • 4 – Column Wash
  • Make sure ethanol solution has passed through the column, if not spin at 13,000rpm for 1 minute
  • Discard any flow through and reinsert column into collection tube
  • Repeat steps twice using 400μl Wash Solution A each time and making sure that all solution has passed through
  • Spin the column dry for 2 minutes at 13,000rpm once washing steps are complete
  • Discard the collection tube

    • 5 – RNA Elution
  • Place column into a fresh 1.7mL Elution tube and add 50μl of Elution Solution A
  • Centrifuge for 2 minutes at 2,000rpm, then for 1 minute at 14,000rpm
  • Check all 50μl of elution solution has passed through, if not spin again for 1 minute at 13,000rpm
  • RNA concentration can then be determined via nanodropping

    • Reverse Transcription Reaction (performed according to manufacturer’s instructions - qPCRBIO cDNA Synthesis Kit)
    1 – Mastermix for RT +ve Samples
  • 4μl 5x cDNA Synthesis Mix
  • 1μl 20x RTase
  • X μl Total RNA (up to 0.4μg)
  • Make up with autoclaved H2O to 20μl
  • Total Reaction Volume = 20μl

    • 2 – Mastermix for RT -ve Samples
  • 4μl 5x cDNA Synthesis Mix
  • X μl Total RNA (up to 0.4μg)
  • Make up with autoclaved H2O to 20μl
  • Total Reaction Volume = 20μl
    • 3 – Incubation and Enzyme Denaturation
  • Incubate at 42°C for 30 minutes
  • Incubate at 85°C for 30 minutes
    • Run agarose gel electrophoresis with appropriate primer pairs