Protocols
Construction protocols
Heat shock protocol (transformation)
o Thaw competent cells on ice. Pipet 2 µl of DNA per 100 µl of competent cells.
o Incubate on ice for 30 minutes.
o Heat shock the mixture of competent bacteria cells and DNA by placing on
thermoblock or waterbath at 42°C for 45 seconds and then immediately place back
on ice for 5 minutes.
o Add 800 µL LB media to transformed cells. Incubate on 37°C for 30 min with
agitation.
Plasmid DNA purification using centrifuges
o Harvest your bacterial culture (3-10 ml) by centrifugation at 3000 rpm for 3 minutes
at room temperature. Decant the supernatant and remove remaining medium. All
other centrifugations should be carried out at 14 000 rpm at room temperature.
o Resuspend the pelleted cells in 250 µL of Resuspension Solution. The bacteria should
be resuspended completely by vortexing or pipetting up and down until no cells
clumps remain.
o Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times
until the solution becomes viscous and slightly clear.
o Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by
inverting the tube 4-6 times.
o Centrifugate the mixture for 5 min.
o Transfer the supernatant to the supplied GeneJET spin column by decanting ot
pipetting. Avoid disturbing or transfering the white precipitate. Bag with Gene JET
Spin Columns must be closed tightly after each use.
o Centrifuge the supernatant for 1 min. The flow-through should be discarded and the
column should be placed back into the same collection tube.
o Add 500 µL of the Wash Solution to the GeneJET spin column, centrifuge for 1
minute and discard the flow through, placing the column back into the same
collection tube. Repeat.
o Centrifugate the columns for additional 1 min to remove residual Wash Soulution.
This step is essential to avoid residual ethanol in plasmid preps.
o Transfer the GeneJET spin column should into a fresh 1,5 ml microcentrifuge tube.
Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to
elute the plasmid DNA. The membrane must not be contacted with the pipette tip.
Incubated for 2 minutes at room temperature and centrifugate for 2 minutes.
Repeat.
o Discard the column and store the purified plasmid DNA at -20°C.
Making Gel for gel electrophoresis
o Choose the concentration of gel you want to use (e.g. 0.7%, 1%, 2%...)
o Measure out the relevant percentage of agarose powder (e.g. 2g of agarose for
100ml of 2% gel).
o Add the relevant volume of 1xTAE buffer (e.g. 100ml of 1xTAE for 2g of agarose for
2% gel)
o Microwave on low for 1-3 minutes, constantly taking it out every so often to swirl
and mix it. Keep microwaving until clear. Make sure it is completely clear.
o Cool to a temperature that is still hot but still can be held in your hand. If the gel gets
too cold and starts to harden, start microwaving again until clear.
o Add 3µl of ethidium bromide for every 100ml of gel after cooled to the correct
temperature. Mix it well by swirling.
o Pour it onto the gel tray with the combs in place.
o Wait 20-30 minutes until the gel completely solidifies.
o Place the agarose gel into electrophoresis unit.
o Add loading buffer to each of your samples.
o Fill the electophoresis unit with 1xTAE until the gel is completely covered.
o Load 7 µl of molecular weight ladder into the first well and load your samples in
additional wells.
o Run the gel at 90V-120V and 400 mA until the dye line is half the way down the gel
(30-45 minutes).
o View the gel in a device with UV light.
DNA extraction from sludge
o Add 500 µL (0,2g) of sludge to a microcentrifuge tube and centrifugate at 14,000 rpm
10 minutes.
o Lyse the sludge pellet by adding equal volume of sterile glass beads and 500µL of
extraction buffer hexadecyltrimethyl ammonium bromide (CTAB).
o Mix the suspension by vortexing for 1minute.
o Add 500 µL of phenol:chloroform:isoamyl alcohol (25:24:1) to the mixure.
o Mix the suspension by vortexing for 1 minute and incubate on ice for 1 minute
(keeping on ice prevents cells from degradation).. Repeat this step three times.
o Centrifugate the mixture at 14,000 rpm for 10 minutes at 4°C. Transfer the supernatant
to a new microcentrifuge tube.
o Repeated the phenol extraction with 500 µL of phenol:chloroform:isoamylalcohol
(24:1) and centrifugate at 14,000 rpm for 10 minutes at 4°C.
o Precipitate the DNA with 0,1 volume of 3M NaOAc (sodium acetate) pH 5.5 and 0.6
volume of isopropanol at -20°C for 30 minutes.
o Wash the DNA pellet with 70% ice cold ethanol. Air dry and resuspend in 30 µL of TE
buffer containing 0.002% RNase.
o The extracted DNA is used for following PCR amplification.
DNA gel extraction
o Excise gel slice containing the DNA fragment using a clean scalpel or razor blade and
place into a clean 1,5 mL tube.
o Add 500 µL of Binding Buffer.
o Incubate the mixture on 60 °C for 10 minutes until the gel slice is completely
dissolved. Vortex the gel mixture briefly before loading on the column.
o Transfer up to 800 µL of the solubilized gel solution to the GeneJET purification
column. Centrifugate for 1 minute. Discard the flow through and place the column
back in the same collection tube.
o Add 700 µL of Wash Buffer. Centrifugate for 1 minute. Discard the flow-through and
place the column back into the same collection tube .
o Centrifugate the empty GeneJET purification column for 1 minute to remove residual
Wash Buffer.
o Transfer the GeneJET purification column into fresh 1,5 mL microcentrifuge tube.
Add 10 µL of Elution Buffer to the centre of purification column membrane.
Centrifugate the mixture for 1 minute.
SDS –page
o Prepare the separating gel.
o Set the casting frames on thje casting clams and pipet the
appropriate amount of gel into the gap between the glass plates. Fill with isopropanol to the top.
o Wait until the gel has hardened (20 minutes).
o Prepare the stacking gel.
o Discard the isopropanol and fill with stacking gel to the top.
o Insert the comb making sure there is no air trapped underneath.
o Wait until the gel has hardened (20 minutes).
o Remove the comb and put the gels into the chamber. Fill with 1xSDS buffer to the appropriate line.
o Load 7 µl of protein marker in the first well and your samples in the additional wells.
o Run at appropriate power (35 mA for 1 gel, 70 mA for 2 gels) for 35 minutes or until the dye line reaches the bottom of the gel.
o Place the gels into the coloring fluid for 30 minutes.
o Place the gels into the fluid for discaloration for 30 minutes.
Transferring the protein from the gel to the membrane ( Western blot)
o Prepare your materials: sponges, filters, gel and membrane.
o Activate the membrane by covering in methanol and rince the membrane, sponges, filters and gel with transfer buffer.
o Prepare the stack as follows (loading on the anode): sponge, 3 filters, gel, membrane, 3 filters, sponge. Make sure no air obubbles are traped in the stack.
o Place the cassette in the transfer tank togheter with an ice pack. Fill the tank with transfer buffer to the appropriate line.
o Run at 200 mA for 1,5 hours.
o Store the membrane in 5% milk for 1 hour at room temperature.
Antibody incubation:
o Take the membrane from the milk and wash it with PBS buffer.
o Mix 10 ml of PBS with 5 µl of primary antibodies and cover the membrane, shaking it for 1 hour and turning it regularly.
o Remove the mixture from the membrane and wash the membrane with PBS.
o Mix 10 ml of PBS with 2µl of secondary antibodies and cover the membrane, shaking it for 1 hour and turning it regularly.
o Wash the membrane with PBS.
o Mix 7 µg DAB, 10 mL PBS and 20 µL H2O2. Cover the membrane and wait a few seconds until the colour appears.
o Remove the membrane and dry it.
Protein expression with immobilized-nicel affinity chromatography (V= 400 ml)
o Centrifuge the sample for 10 minutes at 5000 rcf and room temperature.
o Discard the supernatant and resuspend the pellet in Binding buffer.
o Sonicate the sample for 5 minutes while keeping it on ice. Repeat 3 times.
o Centrifuge for 10 minutes at 30 000 rcf and room temperature.
o Transfer the supernatant to a clean centrifuge tube and discart the pellet.
o Take a sample of the supernatant and run it trought nicel affinity column. Take a sample of the flow trought.
o Run the Elution buffer throught the column and collect it.
o Complete and SDS page with extracted proteins.
Protocol for expression of CtfA, CtfB and BdhB
Escherichia coli DH5α bacteria were transformed with the expression plasmids and grown in shaker flasks at 37 °C in LBC medium overnight. 1 ml samples were taken for analysis. Cells were collected by centrifugation at 6000g for 10 min and resuspended in 100 µl of H20. 10 µl samples were taken and 5 µl of SDS-PAGE loading buffer was added. Samples were cooked at 100 °C for 10 min and resolved on 12% SDS-PAGE gel.
Protocol for isolation of laccases
Escherichia coli BL21(DE3) bacteria were transformed with the expression plasmids (BBa_K863005 and BBa_K863010) and grown in 10 ml at 37 °C in LBC medium overnight. To express both recombinant proteins, 10 ml of overnight cultures shaker cultures were grown at 37 °C in LB broth supplemented with 30 µg/ml chloramphenicol and shaking with 225 rpm. Expression of the recombinant protein was induced by addition of IPTG to a final concentration of 1 mM, when the cell density reached an OD600 of 0.8. After induction, cells were grown for 5 h and then collected by centrifugation at 6000g for 10 min. The cell pellet collected from 400 ml of bacterial culture was resuspended in 20 ml of resuspension buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole) and sonified 3 × 6 min on ice. Following centrifugation at 30 000 × g for 10 min to remove insoluble debris, the supernatant was applied to a Ni-NTA Superflow Cartridge (Qiagen) connected to ÄKTA FPLC system, washed with the resuspension buffer and eluted in the same buffer, but containing 250 mM imidazole. The peak fractions were collected and 15 µl of each fraction was collected and resolved on 12 % SDS-PAGE.