Team:Cardiff Wales/protocols




Our Protocols


Liquid Broths

  • Fill a container with 400ml distilled water
  • Add 25 g/L of LB broth to the distilled water
  • Swirl the container with the water and LB broth together so that the powder has nearly dissolved
  • Autoclave the container and store in fridge at 4 °C until LB broth is required

Agar Plates (8)

  • Fill a container with 200 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 200 µl of antibiotic (Amp, Chl, Kan etc.) to the autoclaved container along with 100 µl X-gal and 100 µl IPTG
  • Distributed finalised mixture equally between 8 petri dishes, making sure all the plates are completely covered and leave to set
  • Once set, store the plates at 4 °C until required.

Transformation of E. coli cells

  • To a 1.5 ml Eppendorf tube, add 50 µl of competent cells to 10ul of DNA and leave on ice for 10 minutes. Heat shock at 42 °C for 1 min before putting the cells back on ice for 5 min. Following this, add 1 ml LB broth with no antibiotic before shaking in the incubator 1 hour at 37 °C. On a chloramphenicol, X-gal and IPTG plate, add 5-10 glass beads with 200 ul of cells and DNA from incubation. Shake the plates until the cells and DNA are equally distributed across the plate. Remove the beads and incubate the plate at 37°C overnight.
  • If the colour of the colonies is indecipherable after incubation, place in 4 °C fridge until colour develops.

Growing Colonies

  • From plate showing successfully transformed white colonies, pick the appropriate colonies using a pipette tip and place into 5ml LB broth with suitable antibiotic (Amp, Chl, Kan etc.) Resuspend the colony in the LB broth and antibiotic before vortexing for 10 seconds. Incubate tubes at 37°C in the shaking machine overnight.

QIAprep® Spin Miniprep Kit

  • Place Buffer EB (for step 10) into 55 °C water bath. Pellet 1-5ml bacterial overnight culture by centrifugation at 13,000 rpm for 3 min at room temperature (15-25 °C)
  • Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
  • Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min
  • Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  • Centrifuge for 10 min at 13000 rpm in a table-top microcentrifuge
  • Apply 800 µl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60s and discard the flow-through
  • Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60s and discard the flow-through
  • Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60s and discard the flow-through
  • Centrifuge for 1 min to remove residual wash buffer. After this, tap the QIAprep 2.0 spin column on the bench before centrifuging again for 1 min
  • 10) Place the QIAprep 2.0 spin column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30-50 µl Buffer EB to the centre of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min

Jena Bioscience: Fast-n-Easy Plasmid Mini-prep Kit

  • Harvest the bacterial cell culture (1-3 ml) by centrifugation
  • Resuspend pelleted bacterial cells in 300 µl lysis buffer by pipetting
  • Add 300 µl of Neutralization buffer (containing RNase A) to sample and mix gently by inverting the tube 4-6 times (do not vortex!)
  • Centrifuge at 10,000 g for 5 min at room temperature in a microcentrifuge
  • The colour of the binding mixture should change to bright yellow indicating a pH of 7.5 required for optimal DNA binding. An orange or violet colour shows a pH >7.5 and indicates and inefficient DNA absorption. In this case, it is recommended to adjust the pH of the mixture by addition of a small volume of 3 M sodium acetate, pH 5.0 before proceeding
  • Place a binding column into a 2 ml collection tube
  • Add 100 µl of Activation buffer into a Binding Column
  • Centrifuge at 10,000 g for 30 sec in a microcentrifuge
  • Apply the supernatant from step 2 into the activated Binding Column by decanting or pipetting
  • Centrifuge at 10,000 g for 30 sec
  • Discard the flow-through
  • Place the DNA loaded Binding Column into the used 2 ml tube
  • Apply 500 µl of Washing Buffer (containing Ethanol) to the Binding Column
  • Centrifuge at 10,000 g for 30 sec and discard the flow-through
  • Add 700 µl of Washing Buffer to the Binding Column
  • Centrifuge at 10,000 g for 30 sec and discard the flow-through
  • Centrifuge again for 2 min to remove residual Washing Buffer
  • Place the Binding Column into a clean 1.5 ml microtube
  • Add 30-50 µl Elution Buffer or dd-water to the centre of the column membrane
  • Incubate for 1 min at room temperature
  • Centrifuge at 10,000 g for 1 min to elute DNA

PCR

  • For a 20 µl PCR reaction, add 10 µl PCR Super Master Mix/Taq Buffer, 1 µl forward primer, 1 µl of backward primer, 6 µl autoclaved water and 2 µl Mini-prepped DNA, ensuring PCR products in are in suitable PCR tubes
  • Place the PCR tubes into the PCR machine and run PCR at a temperature and duration appropriate for the primers to work effectively
  • Once finished, add 6 µl of PCR product to individual wells on a gel (See Agarose Gel Electrophoresis)

Agarose Gel Electrophoresis

  • Add 5 ml of 50x TAE (Tris-acetate-EDTA) buffer to 245 ml of distilled water
  • Take 100 ml of 2% TAE Stock from step 1 and add to a 250 ml conical flask
  • Add 1 g Agarose to 100 ml TAE buffer and microwave for 1 min 50 sec, making sure all agarose has dissolved
  • Cool solution before adding 5 µl Safe View and mix before pouring into gel cassette. Add appropriate number of combs to the cassette, depending on the number of PCR reactions
  • Leave 1% agarose gel to set for 30 min before removing the comb(s). Place set gel into the gel electrophoresis tank and add remaining 2% TAE stock to the tank, making sure the gel is covered by 2% TAE buffer
  • Add 6 µl hyperladder suitable for the DNA length to the first well and add the remaining PCR reaction mixtures to the wells
  • Set the voltage to 120V and run the gel for 30 minutes
  • Once fully run, turn off the voltage and view the DNA bands using a UV illuminator

Transformation of Agrobacterium

  • Add 100 µl Agrobacterium to 5 µl DNA to a 1.5 ml Eppendorf tube and leave on ice for 10 min
  • Freeze in liquid nitrogen for 5 min before incubating at 37 °C for 5 min
  • Place the mixture back in ice for another 5 min before adding 1 ml LB broth with no antibiotic
  • Shake mixture at 28 °C for 2 hr before plating onto LK-Rif plates. Incubate these plates at 28 °C and leave for 2 days

Preparation of EZ protein extraction buffer (30ml)

  • 125 mM Tris-HCl pH 8.8
  • 10% glycerol
  • 50 mM Na2S2O5 (Sodium metabisulfite)
  • Add distilled water to make up to 30 ml

Preparation of Buffer Z (10ml)

  • 125 mM Tris-HCl pH 6.8
  • 10% glycerol
  • 22% BME
  • 0.001% bromphenol blue
  • Add distilled water to make up to 10ml

Preparation of resolving gel

  • 4ml Water
  • 2.5ml of 1.5 M Tris pH 8.8
  • 3.3ml of 30% Acrylamide
  • 100 µl of 10% SDS
  • 100 µl of 100% APS
  • 5 µl of TEMED

Preparation of a stacking gel

  • 5.9ml Water
  • 2.6ml of 1M Tris
  • 1.7ml of 30% Acrylamide
  • 100 µl of 10% SDS
  • 100 µl of 100% APS
  • 5 µl of TEMED

Preparation of the running buffer (1L)

  • 14.4g of Glycine
  • 3g Tris
  • 10ml 10% SDS

Protein purification from infiltrated leaves

  • Grow agrobacteria containing pGB:35s-TSHH-NosT in 5 ml of LB + Kan + Rif in a 50 ml tube
  • Incubate at 28 °C overnight
  • Overnight bacterial cultures of A.tumefaciens containing the plasmid of interest are harvested by centrifugation (20 min/4 °C/4000 rpm)
  • Resuspend agrobacterium in infiltration buffer (MES, Acetosyringone) to OD600: 0.5 using 10 mM MES (pH 5.6), 10 mM MgCl2 and 150µl/ml acetosyringone
  • Add 1 ml MES/MgCl2 buffer and 1.5 µl acetosyringone and measure the concentration. Continuously adding these volumes of buffer and acetosyringone until an OD600: 0.5 is reached
  • Once at OD600: 0.5, incubate mixture at room temperature for 2 hr
  • Hand-infiltrate into silenced N.benthamiana into at least 5 tobacco leaves with a 1 ml needless syringe
  • To a pestle and mortar, add liquid nitrogen and leave till it evaporates and repeat
  • Taking leaf samples from the infiltrated tobacco, place into pestle and mortar and add liquid nitrogen and leave until evaporation occurs
  • Grind up the leaves into a fine powder and add more liquid nitrogen
  • Separately, freeze a 1.5 ml Eppendorf tube in liquid nitrogen before adding the grinded leaves to the Eppendorf tube
  • Add 500 µl of EZ buffer stock (EZ buffer + protease inhibitor) to the tube with the leaves. This is the TOTAL sample
  • In a separate Eppendorf, add 10 µl Buffer Z to 40 µl of the total sample
  • To the total sample containing the leaves, add 20 µl Nickel beads/100 µl of EZ buffer and mix at 4 °C for 3 hr
  • Collect the beads with the magnet. Remove 40 µl of supernatant and 10 µl Buffer Z. this is the Elute 1 sample
  • Wash beads with 500 µl of EZ buffer (no SDS) + 0.2% Tween and mix for 15 min at 4 °C
  • Collect beads and remove 40 µl supernatant and 10 µl Buffer Z. This is ELUTE2
  • Repeat the wash twice. There is no need for additional ELUTE samples
  • After second wash, collect beads and remove 40 µl supernatant and add 10 µl Buffer Z. This is ELUTE3 sample
  • Remove the remaining supernatant and add 40 µl EZ buffer with 10 µl Buffer Z. This is BEADS sample
  • Samples containing Buffer Z must be frozen at -20 °C overnight

Running a PAGE gel

  • Transfer samples from Day Three to a 0.5 ml Eppendorf tube and boil for 10 min in PCR machine
  • Collect the supernatant and allow samples to cool
  • Load 2x PAGE gels with a repeated set of 25 µl samples. (TOTAL, ELUTE1, ELUT2, ELUTE3, BEADS)
  • Run gel at 150V until buffer line reaches bottom of the gel
  • Separate gel and add 1x to Coomassie stain and leave to stain for 1 hr
  • Add de-staining solution to the samples once staining has finished and leave overnight
  • Samples are now ready to analyse

Level 0 digestion and ligation (into pSB1C3)

  • 100ng of plasmid DNA (PSB1C3g) was added to each reaction tube
  • Insert DNA was added in a 2:1 ratio to the plasmid DNA in each separate tube
  • To each reaction mixture 0.5µl BsmB1 and 2µl BSA were added and water was added to make each reaction up to 20µl
  • The digest was performed at 55°C for 2 hours
  • The enzymes were then denatured at 80°C for 15 minutes
  • To each reaction tube 0.5µl T4 ligase and 2µl T4 ligase buffer were added
  • The ligation reaction proceeded at 16°C for 2 hours and was left at 4°C overnight if necessary

Level 1 digestion and ligation (into PGB-A2)

  • 100ng of the plasmid DNA (PGB-A2) was added to each reaction tube
  • All three inserts were added to the tubes in a 3:1 ratio to the plasmid
  • To each reaction mixture 2µl BSA1 and 2µl NEB cutsmart buffer was added and water was added to make each reaction up to 20µl
  • The digest was performed at 37°C for 2-3 hours
  • The enzymes were then denatured at 80°C for 15 minutes
  • To each reaction tube 0.5µl T4 ligase and 2µl T4 ligase buffer were added
  • The ligation reaction proceeded at 16°C for 2 hours and was left at 4°C overnight if necessary

QIAquick Gel Extraction

  • From the gel, cut the correct bands under a blue light with an orange filter (or alternative)
  • Place each cut band into a sterile Eppendorf tube before adding 500 µl QG to each tube
  • Melt the extracted DNA at 50 °C for 10 min until all gel/DNA has melted
  • Add 166 µl isopropanol and mix by inverting 4-6 times
  • Add 1 ml of this solution to a clean spin column and centrifuge for 1 min at 13,000 rpm
  • Discard the flow from the spin column before adding 750 µl PE buffer
  • Centrifuge for 1 min at 13,000 rpm before discarding the flow through
  • Dry the spin column for 1’ at 13,000 rpm. Repeat this step
  • Transfer column into a new microcentrifuge tube and add 30 µl elution buffer. Leave to stand for 1 min before centrifuging for 1 min

Luciferin assay

  • To make 100 ml of buffer:
  • 50 ml of 100 mM Mes pH5.6 – ( Gives 50mM Mes)
  • 0.5 ml DMSO – (gives 0.5% DMSO)
  • 0.5 g Glucose – (gives 0.5% Glucose)
  • 0.2 ml EDTA – (gives 1mM EDTA)
  • 49 ml H2O
  • Add 200 μl of buffer to each well of a black 96 well plate
  • Use the grade 2 cork borer to cut leaf discs out of the leaves to be tested and place one disc into each well of the 96 well plate
  • Add 2 or 4 μl of Luciferin, giving 0.5 or 1 mM Luciferin solutions to each of the wells. Image within 1 hour
  • Extract the data (ph/s/cm2/sr) from the imager to excel to be manipulated

Making Competent E.coli

  • Inoculate 5 ml of Liquid Broth with E.coli in a 50 ml falcon tube and grow overnight at 37 ̊C with shaking
  • Make up 800 ml of LB Broth (20 g) in a 2 litre conical flask and autoclave overnight
  • From step 4 onwards everything is to be done on ice/at 4 ̊C
  • Dilute the overnight culture by transferring it to the 800 ml of LB Broth and place the solution back in the 37 ̊C shaker for approximately 2-3 hours/or until the absorbance at 650 nm is between 0.2 and 0.4
  • Chill the culture in an ice water bath for 10 minutes after transferring to pre-chilled 50 ml Falcon tubes
  • Harvest the cells by centrifugation at 4000 rpm and 4 ̊C for 10 minutes
  • Pour off the supernatant and re-suspend, on ice, in ¼ culture volume (200 ml) 100 mM MgCl2 and leave on ice for 5 minutes
  • Harvest the cells again by centrifugation at 4000 rpm and 4 ̊C for 10 minutes
  • Pour off the supernatant and re-suspend, on ice, in ¼ culture volume (50 ml) 100 mM CaCl2 and leave on ice for 20 minutes
  • Centrifuge at 4000 rpm and 4 ̊C for 10 minutes
  • Re-suspend in 2.5 ml culture volume of 100 mM CaCl2, 15% glycerol, on ice
  • Pipette 100 μl aliquots of liquid culture into pre-chilled Eppendorf’s on ice and freeze in liquid nitrogen before storing at -80 ̊C

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