Team:Macquarie Australia/Experiments
Experiments & Protocols
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This page is for descriptions of the experiments, research and protocols we used in our iGEM project.
Protocols
Making competent cells
1.
Using a sterile
plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate.
Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22oC,
200-250rpm.
2.
A600 should be
0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~
0.5.
3. Remove the flask from the incubator and place on ice
for 10 minutes.
4. Transfer the culture to a 15mL centrifuge tube and
spin at 2500 x g for 10 min at 4oC
5. Pour off and discard the supernatant, and immediately
place the tube on ice.
6. Resuspend your cells in 1mL of ice-cold TB buffer,
make sure there are no clumps of cells left, but also treat your cells gently
and keep them cold.
7. Add ice-cold TB buffer to bring volume up to 1/5th of
the original culture volume (~30mL in this case). Mix the tube by gently
inverting 3 times.
8. Incubate the tube on ice for 10 minutes.
9. Centrifuge at 2,500 x g for 7
minutes at 4oC, discard the supernatant.
10.
Gently resuspend
the cells in ~1/20th of the original culture volume of ice-cold TB buffer.
NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th
of original volume.
11.
Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension,
keeping the remainder on ice in the 15mL tube.
12.
Add 70µl of DMSO
to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
13.
Aliquot 100µl of
the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the
tubes with: Date – Strain. Snap freeze with
liquid nitrogen.
14.
Repeat step 11-13
for the rest of your cell suspension in step 10. Store cells at -80oC
TB BUFFER
● Ingredients: 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl.
● Methods: All components (except for MnCl2-4H2O) were mixed and
dissolved in 500 mL of water and pH adjusted to 6.7
with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL
of water, mixed and solution adjusted to 1 L. Sterilisation via filtration
followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.
EDTA BUFFER
● Ingredients: 37.22g EDTA solid, 180 mL
of water and pH adjusted to 8.0 using 10M NaOH.
● Methods: Components were combined then pH adjusted.
TAE BUFFER
● Ingredients: 121.2g Tris base (dissolved
in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0).
● Methods: A total volume of 500 mL
was made up as a 50x stock solution using all components
Making agarose gel
Preparing the Gel
1. Mix 1g of agarose powder with 100ml of 1x TAE buffer
and heat for 1minute or until all agarose is dissolved.
2.
Wait until it has
cooled (not set), and add 1ul of GelRed into the
mixture.
3.
Pour the solution
into a cast with an appropriate comb.
4.
Leave to set.
Running the Gel
1. Mix 1ul of 1kbp DNA ladder with 6ul of loading dye (bromophenol blue) and 4ul of 1x TAE buffer (total 6ul) and
load onto first well
2.
Mix 5ul of PCR
products with 1ul of loading dye and load onto wells.
3.
Run gel at 90V
for 45minutes approximately
4.
Photograph gels
under UV light
Composite part ligation
The assembly of composite parts from
two existing BioBricks i.e. BioBrick
plasmid A (parent vector) and BioBrick plasmid B
(gene to be inserted) was performed through a restriction digest/ligation
protocol.
1.
200ng of each BioBrick plasmid was digested as follows: Plasmid BioBrick A with NEB restriction enzymes SpeI
and PstI; plasmid BioBrick B with XbaI and PstI, according to supplier protocol. (1hr @37oC,
then 20mins @80oC).
2.
1µL of Fast alkaline phosphatase (Thermo
Scientific) was added to reaction tube of plasmid BioBrick
A to dephosphorylate the gene fragments and prevent
re-ligation of the parent vector. Reaction tubes were incubated at 37oC
for 60 mins, followed by a deactivation step at 80oC for 20 mins.
3.
Ligation was then
performed with an insert to vector molar ratio of 3:1. 1µL of T4 ligase (NEB)
was added to the mix for ligation, according to supplier protocol. Ligation
reactions were performed at 37oC for 60 mins and kept on ice for
transformation into chemical competent cells.
MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS
1. Coommassie de-stain gels: by washing briefly with 200µL NH4HCO3
(Solution A) to make sure gel pieces are at the correct pH.
2.
Add 200µL 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) into each
well. Vortex to mix and incubate for 10 minutes. Remove liquid.
3.
Repeat step 2.
Gel pieces should be clear at this stage. If they are still blue, repeat as
necessary until color is gone.
4.
Wash for 5 min
with 50µL of 100% Acetonitrile (Solution C) to
dehydrate gel pieces. Vortex during incubation.
5.
Remove Acetonitrile, then let air-dry for
10 min. The gel pieces should be noticeably shrunken and probably white.
6.
Reduction and
Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D).
Let proteins reduced for 45-60 min in 37oC incubator.
7.
Remove DTT solution
and add 50µL of 55mM iodoacetamide in 50mM NH4HCO3
(Solution E). Incubate for 45 min in dark place at room temperature.
8.
Remove
iodoacetamide, discard.
9.
Wash gel pieces
with 200ul of NH4HCO3 (Solution A) for 5 min with vortexing.
before adding 100ul of 100% Acetonitrile
(Solution C).
10.
Remove
liquid after 5 min, discard.
11.
Wash gel pieces
with 50ul 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200ul 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) for 10min
12.
Dehydrate with
100 ul 100% Acetonitrile
(Solution C) for 5 min as above.
13.
Remove remaining
liquid and let the gel dried.
14.
Trypsin Digestion: Prepare trypsin
mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate.
15.
Add 30µL (or more
if required) of mixed trypsin solution to cover the
gel pieces.
16.
Allow 30 min for
gel rehydration at 4oC (on ice).
17.
Digest overnight
at 37oC.
18.
Peptide
Extraction: Transfer the digest solution supernatant (if any) into clean 0.65ml
Eppendorf tubes.
19.
To the gel
pieces, add 50 µL of 50% acetonitrile / 2% formic acid,
incubate 30 min. Spin, remove supernatant and combine with initial digest
solution supernatant. Please note that total volume may vary depending on the
gel sizes.
20.
Vortex the
extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume
is less than 10ul, use 2% formic acid to bring the volume up to 10 µL.
21.
Spin at 14,000
rpm for 30 min to remove any micro particulates.
22.
Transfer the
supernatant to a fresh 0.65ml eppendorf tube for
storage at 4oC fridge OR directly into PCR plate for Mass spec for
analysis.
Reagents
● Buffer 100mM Ammonium
Bicarbonate
○ ADD 0.78 g ammonium bicarbonate to 100ml ddH20. Fill
first medium size reservoir.
● Reduction: - Dithiothreitol (DTT)
○ Add 15 mL of 100 mM ammonium bicarbonate to 23.1 mg of DTT to give a solution
of 10 mM DTT.
● Alkylation - Iodoacetamide(DTT)
○ Add 15 mL of 100 mM ammonium bicarbonate to 153 mg of iodoacetamide
to give a solution of 55 mM iodoacetamide
- 15 mL of each is enough for up to 96 samples.
● Peptide Extraction Solution - 2% Formic acid/ 50% Acetonitrile solution
○ Add 2000µL of formic acid and 50 mL
of acetonitrile to 75 mL of
ddH2O. 15 mL is enough for up to 96 samples.
● Dehydration - Acetonitrile
○