Team:Bristol/Protocol

These are the protocols we used in the lab! We refer to these in our Lab Notebook.

PCR (with Hifi/Ultra DNA polymerase)

  • Primer concentration: 10 pmol/µl
  • Template concentration: 1 ng/µl unless otherwise stated.
  • Samples were prepared in the order shown in the following table:

DNA gel electrophoresis

  • For a large 20 well 1% agarose gel:
  • • Mix 100 ml 1X TAE buffer and 1g fine agarose in a conical flask.
  • • Microwave on high power until bubbles appear. Mix, then microwave until bubbles form a second time and the solution is transparent.
  • • Cool under cold water tap until solution is no longer steaming.
  • • Add 3 µl ethidium bromide.
  • • Pour agarose solution into gel mould, and allow to set for approx. 20 mins. When set, remove comb.
  • • Sample preparation: mix 5 µl of PCR product with 2 µl loading dye.
  • • Load 6 µl of ladder into the first well, and 6 µl of PCR samples into subsequent wells.
  • • Run the gel for 50 minutes at 100V, 1000 mA and 150 W.

Tip: Use half quantities for smaller 10-12 well gels

Purification of PCR products

  • We used a Qiagen PCR purification kit, making some small adjustments to the instructions given:
  • • Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix (e.g. 225 µl buffer, 45 µl sample).
  • • Place a QIAquick spin column in a 2 ml collection tube.
  • • To bind DNA, apply the sample to the QIAquick column and centrifuge for 60 s.
  • • Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.
  • • To wash, add 750 µl Buffer PE to the QIAquick column and centrifuge for 60 s.
  • • Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.
  • • Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
  • • To elute DNA, add 50 µl hot water (70°C) to the centre of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

Gibson assembly

  • • Calculate fragment and vector backbone volumes based on fragment concentration, using calculator provided on manual. Final volumes should contain 50-100 ng of vector with 2-3 fold of excess inserts (may be up to 5 times depending on length of insert).
  • • Incubate samples at 50°C for 15-60 minutes (can incubate for up to 4 hours if ligating 4+ fragments).
  • • Transform competent cells using transformation protocol listed or store product at -20°C.

Competent cell transformation

  • • Thaw competent cells on ice for 15-20 minutes.
  • • Add 2 µl of your DNA to the cells and flick gently to mix. Place back on ice for at least 20 minutes, or up to 4 hours.
  • • Heat shock cells at 42°C for 45 seconds then place back on ice for approximately 2 minutes.
  • • Add 200 µl of SOC media and flick gently to mix.
  • • Place cells in a incubator at 37°C for 1-2 hours to allow expression of antibiotic resistance gene.
  • • Remove cells from incubator, spread on agar plate containing appropriate antibiotic and incubate overnight at 37°C.

Making LB/agar plates

  • • Weigh out 5g of LB powder and 3.75g of agar and add to 250 ml of deionized water.
  • • Use magnetic flea to mix until the agar is dissolved.
  • • Stick autoclave tape to bottle and place bottle in autoclave ensuring lid is left loose and there is sufficient water (magnetic flea does not have to be removed).
  • • Autoclave then mix and allow to cool until approximately 50°C.
  • • Once cooled, add 1/1000 dilution (250 µl) of your chosen antibiotic.
  • • Plate under semi-sterile conditions (next to bunsen burner) in smooth motion, until base surface is covered. Leave lid slightly open to minimise condensation, before incubation.

Tip: 250 ml is sufficient to make 8-10 plates.

Gel extraction

  • The Qiagen gel extraction kit we used can be found  here.
  • • Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  • • Weigh the gel slice in a colourless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100 µl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
  • • Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
  • • Add 1 gel volume isopropanol to the sample and mix.
  • • Place a QIAquick spin column in a provided 2 ml collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 µl, load and spin again.
  • • To wash, add 750 µl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
  • • Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  • • To elute DNA, add 50 μl hot water (70°C) to the center of the QIAquick membrane and centrifuge the column for 1 min.

Making glycerol stocks

  • • Pipette 700 µl of cells and 300 µl of sterile glycerol into eppendorf tube and mix well.
  • • Flash freeze in liquid nitrogen and store at -80°C.

Western blotting

Preparing protein extracts:

  • • Start pre-culture in 10 mL LB+antibiotics
  • • Inoculate (1 ml of pre-culture) new cultures at 1/100 in 100 ml LB+antibiotics
  • • Grow until OD=0.5-0.7 (check OD after 2 hours, then every 30 min until induction)
  • • Add inducers (arabinose 1% and/or IPTG 1 mM)
  • • Grow 2 more hours (measure OD)
  • • Centrifuge at 3800g for 10 min
  • • Resuspend the pellets in 1 ml of the BugBuster reagent (Novagen). Transfer to eppendorfs
  • • Incubate 20 min at room temperature with gentle agitation (rotating wheel)
  • • Centrifuge for 10 min at 10000g
  • • Take 75 µl of each sample and add to 25 µl NuPage LDS Sample Buffer (Novex). Boil for 10 min at 95°C in the dry block. Store at -20°C.
  • • Take some of the supernatant for a Lowry assay. Keep the rest of the supernatant at -20°C.

Lowry protein assay (DC protein assay – Biorad):

  • A protein assay is done to ensure you load equal amounts of protein to each well.
  • • Add 20 µl of Reagent S to 1 ml Reagent A
  • • Add 125 µl of S+A to 25 µl sample. Don’t forget to prepare a blank sample with just buffer (BugBuster reagent, no protein)
  • • Vortex, then add 1 ml Reagent B (glass tube)
  • • Incubate on the spinning wheel for 15 min at room temperature
  • • Measure absorbance with the Cary UV-Vis spectrophotometer (Simple Read, 750 nm)

Western blot:

  • • Run a gel: load samples of equivalent protein content (according to Lowry assay): 10-20 µl of protein extract. We used Tris-Glycine 10-12% gels in an Invitrogen tank for 1 hour at 200V. Don’t forget 5 µl marker.
  • • Prepare transfer buffer:
     - 3 g TrizMa
     - 14.4 g Glycine
     - 0.37 g SDS
     - 100 ml methanol
     - Adjust volume to 1 L with water
  • • Soak the following for a few minutes in transfer buffer: 6x fluffy spacers, 2x Whatman paper cut to the size of a gel, 1x nitrocellulose membrane.
  • • Prepare sandwich:
     - 3x fluffy spacers
     - 1x paper
     - Gel
     - Membrane
     - 1x paper
     - 3x fluffy spacers
  • • Transfer for 1 h at 25 V with transfer buffer between the dams, and water outside. Don’t recycle transfer buffer.
  • • Block the membrane with 1 g milk in 20 ml PBS + 0.05% Tween (500 ml 1x PBS+250 µl Tween), overnight (or 1 hour) at 4°C with slow agitation.
  • • Add 2-10 µl of antibody (dilution 1:10000 to 1:2000 for anti-myc) to 20 ml of PBS+0.05% Tween + 1g milk.
  • • Incubate for 1h at room temperature.
  • • Wash with PBS+0.05% Tween 3x for 5 mins then 2x for 1 min.
  • • Incubate 1 minute with 9 ml water+0.5 ml of LumiGlo substrate (reagent A) and 0.5 ml peroxide (reagent B).
  • • Image.

Coomassie Staining (ThermoFisher protocol):

  • • Rinse the gel 3 times for 5 minutes with 100 ml deionized water to remove SDS and buffer salts, which interfere with binding of the dye to the protein.
  • • Stain the gel with enough  SimplyBlue™ SafeStain (20-100 ml) to cover the gel. Stain for 1 hour at room temperature with gentle shaking. Bands will begin to develop within minutes. After incubation, discard the stain.
  • • Wash the gel with 100 ml of water for 1-3 hours. The gel can be left in the water for several days without loss of sensitivity. There is a small amount of dye in the water that is in equilibrium with the dye bound to the protein, so proteins will remain blue.
  • • To obtain the clearest background for photography, perform a second 1 hour wash with 100 ml water.

Cyclic voltammetry

Preparation and operation of potentiostat:

  • The instrument used is a uniscan PG580 (other models available) - ensure this has four leads which will connect to the working, counter and reference electrodes as well as an earthing point such as the chassis of your computer. Cyclic voltammetry can then be carried out using programmes such as UiEChem, which allows control of the scan rate, voltage range, data collection and current sensitivity.
  • • Connect the electrodes and turn on the potentiostat.
  • • To start the software, click on the UiEChem item in the Programs section of the computer’s Start menu. Select Cyclic Voltammogram and click OK.
  • • Record cyclic voltammograms of the electrolyte solution setting Sweep Potential 1 (the initial potential), and Sweep Potential 3 both to 0.100 V, and Sweep Potential 2 to 0.55V at 0.002 V s−1.
  • • Record data at 0.001 V per point, and check the ‘Auto’ box. Click the green arrow on the menu bar to start the potential sweep, and then click File » Preferences and uncheck ‘American’. Confirm that a clear reversible signal is obtained.
  • • If current oscillations are observed in a certain potential range, verify that the current range is set at 10 mA.

Preparation of solutions

  • • Prepare 250 ml of 2.5% LB solution ensuring all LB is dissolved (mix well and use microwave if necessary).
  • • Make 100 mM ammonia solution by diluting ammonia hydroxide solution.

Experiment

  • • Fill a 100 ml beaker with equal volumes of of LB and ammonia solution and place on a stirring plate.
  • • Connect leads from potentiostat to corresponding electrodes using crocodile clips.
  • • Clamp the cell to ensure that it does not topple over and the crocodile clips at the ends of the leads do not touch.
  • • Run the programme on the potentiostat detailed above; repeat this three times minimum to get an estimate for the uncertainty in your peak current.
  • • Repeat protocol using negative control (replace ammonia solution with equal amount of deionized water).