Team:Macquarie Australia/Protocols








Electrophoresis

Gel Preparation

  1. Mix 1g of agarose powder with 100ml of 1x TAE buffer and heat for 1 minute 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 1 x TAE buffer (total 6ul) and load onto first well.
  2. Mix 5ul of PCR products with 1ul of loading dye and load into wells.
  3. Run gel at 90V products with 1ul of loading dye and load onto wells.
  4. Run gel at 90V for 45 minutes approximately.
  5. Photograph gels under UV light.

Manual Trypsin Digest

  1. For Coomassie stained gels, de-stain gels by washing briefly with 200uL NH4HCO3 (Solution A) to make sure gel pieces are at the correct pH.
  2. Add 200uL 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 colour is gone.
  4. Wash for 5 min with 50uL 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 50uL 10mM DTT in 50mM NH4HCO3 (Solution D). Let proteins be reduced for 45-60 min in a 37oC incubator.
  7. Remove DTT solution and add 50uL 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 dry.
  14. Trypsin Digestion: Prepare trypsin mix to final concentration of 13ng/uL in 50mM Ammonium bicarbonate. Add 30uL (or more if required) of mixed trypsin solution to cover the gel pieces. Allow 30 min for gel rehydration at 4oC (on ice). Digest overnight at 37oC.



Peptide Extraction

  1. Transfer the digest solution supernatant (if any) into clean 0.65ml Eppendorf tubes.
  2. To the gel pieces, add 50 uL 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.
  3. Vortex the extracted digests, speed vac to reduce volume to 10 uL. If the remaining volume is less than 10uL, use 2% formic acid to bring the volume up to 10 uL.
  4. Spin at 14,000 rpm for 30 min to remove any micro particulates.
  5. 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.



Trypsin Preparation

  1. Trypsin is prepared by adding 200uL of Resuspension Buffer into one vial containing 20ug of Proteomics grade trypsin.
  2. Add 1.2ml of 50mM NH4HCO3 into each trypsin vial (final concentration of Trypsin is 16ng/uL). Mix thoroughly.
  3. This will provide enough Trypsin for 46 digestions.



Buffers and Reagents

50x TAE Buffer

Tris Base (MW=121.1) 242.4g
EDTA Disodium Salt 18.61g
Glacial Acetic acid 57.1mL

Dissolved Tris base and EDTA in 700mL distilled water, then added glacial acetic acid. Additional water was added to bring final volume up to 1L. Diluted to 1x for use.




50x M (100mL)

Na2HPO4 17.75g
KH2PO4 17g
NH4Cl 13.4g
Na2SO4 3.55g

Made up to 100mL with distilled water. Autoclaved for 15 minutes to sterilise.




ZY Media (400mL)

Tryptone 4g
Yeast Extract 2g

Made up to 400mL with distilled water. Autoclaved for 15 minutes to sterilise.




50x 5052 (100mL)

Glycerol 25g
Glucose 2.5g
Alpha Lactose 10g

Dissolved glycerol in 40mL of distilled water. Dissolved glucose and alpha lactose in 40mL of distilled water. Combined two solutions, adjusted final volume to 100mL. Autoclaved for 15 minutes to sterilise.




TB Buffer (500mL)

KCI(MW=74.5513) 9.3g
CaCl2.2H2O (MW=147.0146) 1g
PIPES (MW=302.4) 1.5g
MnCl2.4H2O 5.44g

Dissolved KCl, PIPES and CaCl2 in 300mL of Milli-Q water. pH adjusted to 6.7 using KOH. Dissolved 5.44g of MnCl2 in 100mL of Milli-Q water. Gradually added MnCl2 to the other solution. Final volume was adjusted to 500mL, filter sterilised with 0.22um filter, then stored at 4°C




HEPES, pH 7.5 (200 mL)

HEPES 47.66g
KOH As required

Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCl, then adjusted final volume to 200mL.




Tris-HCl, pH 8.0 (200ml)

Tris 24.228g
HCl As required

Dissolved Tris in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCl, then adjusted final volume to 200mL.




1M Imidazole, pH 8.0 (400ml)

Imidazole 27.232g
HCl As required

Dissolve imidazole in 75% of total volume of Milli-Q water. Buffered pH to correct levels with HCl, then adjusted final volume to 400mL.




1M EDTA, pH 8.0 (200ml)

EDTA 29.22g
NaOH As required

Dissolved EDTA in 75% of total volume of Milli-Q water while adding NaOH to assist. Buffered pH to correct levels with NaOH, then adjusted final volume to 200mL.




Dithiotheritol 1M (5mL)

DTT 0.7715g

Dissolved DTT in Milli-Q water.




TE Buffer (100ml)

TRIS (1M) pH 8.0 1mL
EDTA(0.5M) pH 8.0 0.2mL

Combined reagents, adjusted final volume to 100mL with Milli-Q water.




Cas9 Buffer (100ml)

HEPES(1M) pH 7.5 2mL
KCl(1M) 15mL

Combined reagents, adjusted final volume to 100mL with Milli-Q water.




CaCl2 1M (100mL)

CaCl2 11.098g
KCl(1M) 15mL

Dissolved CaCl2 in 100mL of Milli-Q water.




Ammonium Bicarbonate 100mM (400mL)

Ammonium Bicarbonate 3.164g

Dissolved ammonium bicarbonate in 400mL of Milli-Q water.




Ammonium Bicarbonate 50mM (200mL)

Ammonium Bicarbonate 0.791g

Dissolved ammonium bicarbonate in 200mL of Milli-Q water.




50% 100mM Ammonium Bicarbonate/50% acetonitrile (200ml)

Ammonium bicarbonate 100mM 100mL
acetonitrile 100mL

Combined reagents in Schott bottle.




Coomassie Destain (1L)

Acetic Acid 100mL

Mixed with 900mL Milli-Q water




Coomassie Fixing Solution (400mL)

Acetic Acid 40mL
Ethanol 200mL

Combined reagents and adjusted final volume to 400mL with Milli-Q water.




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 2000uL of formic acid and 50 mL of acetonitrile to 75 mL of ddH2O. 15 mL is enough for up to 96 samples.




Competent Cells

  1. A sterilized plastic loop was used to gather 10-12 large (2-3mm in diameter) colonies from the plate. Inoculated to 150mL of SOB medium in a 1L flask and grown overnight at 18-22°C, 200-250 rpm.
  2. A600 readings were between 0.2-0.8 when harvested. Cells were in mid log phase with A600 ~ 0.5.
  3. The flask was placed on ice for 10 minutes after being removed from incubator.
  4. Culture was transferred to a 50mL centrifuge tube and spun at 2,500 x g for 10 minutes at 4°C.
  5. Supernatant was discarded and the tube was placed on ice.
  6. Cells were resuspended in 1mL of ice-cold TB buffer, ensuring no clumps of cells remained and kept cold.
  7. Cold TB-buffer was added to bring up volume up to 1/5th of the original culture volume (~30mL) and mixed gently and inverted 3 times.
  8. Tube was incubated on ice for 10 minutes and centrifuged at 2,500 x g for 7 minutes at 4°C and the supernatant was discarded.
  9. Cells were resuspended in ~1/20th of the original culture volume of ice cold TB-buffer.
  10. 930uL of cell suspension was added to pre-chilled 1.5mL Eppendorf tubes.
  11. 70uL of DMSO was added to the 930uL cell suspension and mixed gently while placed on ice.
  12. 100uL of the competent cell/DMSO mixture was added into fresh microcentrifuge tubes, labelled and snapped frozen with liquid nitrogen.
  13. Steps 10-12 was repeated to the remaining for the rest of the suspension from step 9.




Heat Shock Transformation

  1. Obtain competent cells from -80oC.
  2. Defrost gently on ice. 100uL is sufficient for 2 transformations.
  3. Add 1-10uL of plasmid DNA/ ligation mix to each tube. Incubate on ice for 30 min.
  4. Heat shock in 42oC water bath for 30 seconds, then back on ice for 2 min.
  5. Add 200uL of SOC media to each tube, and incubate in the 37oC shaker for 1h.
  6. For each tube of cells, spread 50uL onto one LB plate with appropriate antibiotic, and 100uL onto a second plate, using aseptic technique. Place your plate upside-down in the 37oC incubator.
    7.




Media and Plates


LB Media (400mL)

Tryptone 4g
Yeast Extract 2g
NaCl 4g

Made up to 400mL with Milli-Q Water in a 500mL Schott bottle. Autoclaved for 15 minutes to sterilize.




LB Agar (400mL)

Tryptone 4g
Yeast Extract 2g
NaCl 4g
Bacto Agar 6g

Made up to 400mL with Milli-Q water in a 500mL in a Schott bottle. Autoclaved for 15 minutes to sterilize. 400uL of antibiotic was added once the bottle was cool enough to handle. Plates were poured using antiseptic technique.




SOB Medium (400mL)

Tryptone 8g
Yeast Extract 2g
5M NaCl 0.8mL
1M KCl 1mL
1M MgCl2 4mL
1M MgSO4 4mL

Made up to 400mL with distilled water. Autoclaved for 15 minutes to sterilize.




SOC Medium

Tryptone 8g
Yeast Extract 2g
5M NaCl 0.8mL
1M KCl 1mL
1M MgCl2 4mL
1M MgSO4 4mL
2M D-Glucose 4mL

Made up to 400mL with Milli-Q water. Autoclaved for 15 minutes to sterilize




1% Agarose Gel

DNA Grade Agarose 1g
1x TAE Buffer 100mL
GelRed 4uL

Mixed components in 250mL flask and microwaved until completely dissolved. Cooled slightly before 4uL of GelRed added.




M9 Glycerol Agar

M9 Salts (5x) 100mL
1M MgSO4 2mL
1M CaCl2 50uL
20% Glycerol 10mL
Milli-Q Water 113mL

The next reagents were added to the mixture via filter sterilization:

1% Thiamine HCl 15mL
Cas Amino Acids =10mL

Plates were then prepared using aseptic technique.




M9 Glucose Agar

Agar solution was prepared by autoclaving 7.5g of agar in 250mL of water. The following reagents were combined and autoclaved before being aseptically added to the hot agar:

M9 Salts (5x) 100mL
1M MgSO4 2mL
1M CaCl2 50uL
20% Glucose 10mL
Milli-Q Water 113mL

The next reagents were added to the mixture via filter sterilization:

1% Thiamine HCl 15mL
Cas Amino Acids 10mL

Plates were then prepared using aseptic technique.




Plasmid Prep

  1. Centrifuge at 13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorfs.
  2. Discard supernatant and add 1.9 mL of culture and centrifuge again at 13,200 rpm for 10 min.
  3. Re-suspend pelleted cells in P1 Buffer (250uL).
  4. Add 250uL of P2 buffer and invert 4-6 times (turns homogenous blue).
  5. Add 350uL of N3 and mix by inverting (turns colourless).
  6. Centrifuge at 10min 13,000 rpm to obtain a pellet.
  7. Transfer supernatant in QIA Prep Spin Column by pipetting.
  8. Centrifuge 30-60 sec - discard flow through.
  9. Wash QIA Prep Spin Column with 0.5mL of PB.
  10. Centrifuge for 30-60 seconds - discard flow through.
  11. Wash spin column by adding 0.75 mL PE buffer.
  12. Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1 min.
  13. Place QIA Prep Column in clean Eppendorf.
  14. Elute DNA by adding 30uL of water, stand for 1 min, centrifuge for 1 min.
  15. Plasmid concentration is measured using NanoDrop 2000 spectrophotometer.

Protein Expression


IPTG Induction

  1. Re-transform restriction-digest screened plasmids according to the heat shock transformation protocol.
  2. Screen plates for transformant colonies and place in 5mL of LB broth with appropriate antibiotic concentration at 37oC with shaking until OD600¬ is 2.0 – 3.0.
  3. Take whole volume and place in 45mL of LB broth in a sterile conical flask containing appropriate antibiotic concentration. Incubate at 37oC with shaking until OD600 is 0.5-0.7.
  4. Transfer 1M IPTG in a 1:1000 dilution into flask. Incubate at 15oC with shaking overnight.
  5. Cell pellets were collected through centrifugation at 12,000 rpm for 5 mins.
  6. Cell pellets were resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM Tricene NaOH pH 8.0, 2mM MgCl2, 1mM DTT) and whole lysate was collected through a French Press for functional assays of operon protein products in lysate.

Auto Induction

  1. Start with glycerol stock or freshly transformed plate.
  2. Inoculate 2ml starter culture with a single colony from plate, grow at 37oC with vigorous shaking to OD600 ~ 1.
  3. Transfer to a larger culture (50ml of ZYM-5052 medium in 250ml flask) and grow at room temperature (or 18o C) overnight ~ 200rpm. For small scale, same starter flask can be grown at room temperature for ~24h. Amount of protein expressed peaks at OD600 ~ 8-10.
  4. Spin down 5000 x g for 20 min at 4oC, discard supernatant.
  5. Resuspend pellet in 1/10th of the original volume.
  6. To run on NuPage Bis-Tris gels, make sample mixture which contains: 20 uL resuspended lysate, 20 uL milli-Q water, 16uL NuPage LDS sample buffer, and 5uL 1M DTT.
  7. Boil for 20 min, then spin down max speed for 20 min. Load 10-20uL of supernatant on gels.
  8. Run gel at 150V for 1.5h, then fix with Coomassie Fixing solution for 10 min, stain with Coomassie Stain Solution for 5 min and destain overnight with Destain Solution.

SDS-PAGE


  1. Re-suspend pelleted bacterial cells in 200uL of Milli-Q-water
  2. Transfer 50 uL of suspension into new Eppendorf tubes and combine with 50uL of 2xTruSep sample buffer.
  3. Shear the cells using a Hamilton syringe.
  4. Centrifuge the preparation for 3 minutes at 13,000 rpm.
  5. Load 20 uL of the supernatant into gel.
  6. Conduct electrophoresis at a constant voltage (200V) for 1 hour.
  7. Coomassie Stain for ~30 minutes.

PCR

General mastermix recipe:

      4 uL of 5x phusion buffer
      0.4 uL dNTPs
      0.6 uL of DMSO
      0.2 uL of polymerase
      11.8 uL of water

To this mixture, add:

      1 uL of forward primer
      1 uL of reverse primer
      1 uL of template
      Total volume = 20 uL

PCR settings:

      A general program for PCR is as follows:
      Initial denaturation at 98oC for 30 seconds

    Followed by 30 repeats of:

        Denaturation – 98oC for 10 seconds
        Annealing – 60oC for 10 seconds
        Extension – 72oC for 2 minutes
        Final extension – 72oC for 10 minutes

Standard Assembly


Digestion

1. Digest Upstream Part with EcoRI-HF and SpeI.

Upstream Part Plasmid 400ng
EcoRI-HF 0.5uL
SpeI 0.5uL
10 x NEBuffer CutSmart 1uL
H2O Make up to 10uL

2. Digest the Destination Plasmid that contains the Downstream Part. with EcoRI-HF and XbaI: The Destination Plasmid should also have a different antibiotic resistance marker from the plasmid containing the Upstream Part to avoid the need to purify the Upstream Part.

Downstream Part Plasmid 400ng
EcoRI-HF 0.5uL
XbaI 0.5uL
10 x NEBuffer CutSmart 1uL
H2O Make up to 10uL

3. Incubate all 3 restriction digest reactions at 37°C for 80 min, then heat inactivate at 80°C for 20 minutes.

Ligation

4. Ligate the Upstream and Downstream Parts into the digested Destination Plasmid with a 4:1 insert to vector ratio. The volume of insert required are calculated using.

V = (((X/Y)*4)*A)/B

V= Volume of Insert digest required in uL X= length of Insert (upstream part) in bp
Y= length of Vector (downstream part +backbone) in bp A= Amount of vector used in ng
B= Concentration of Insert Digest in ng/uL

Upstream Part Insert digestion V = (((X/Y)*4)*A)/B
Downstream Part in Destination Plasmid Vector digestion 20 ng
10 x T4 DNA Ligase Buffer 1uL
T4 DNA Ligase 0.5uL
10 x T4 DNA Ligase Buffer 0.5uL
H2O Make up to 10uL

*It is important to note that the combined volume of both digests used in the ligation must not exceed 8uL and is preferably <5uL.

5. Incubate the ligation mix at 37°C for 90 min, then heat inactivate at 80°C for 20 min.




Protochlorophyllide Extraction from Barley


Etiolated barley was prepared by growing the seeds for 8-9 days after germination in the dark.

  1. The stalks of the Yellow sprouts were collected and macerated with liquid nitrogen and soaked in ~600mL acetone for 10 minutes.
  2. The sample was then filtered using filter paper to remove the plant solids and washed with acetone several times or until no more colored pigment is extracted, followed by a final wash with ~400mL of diethyl ether.
  3. The solution was then transferred to a separatory funnel and shaken vigorously.
  4. The lower now colourless aqueous phase was drained and discarded whilst the organic phase was washed several times with roughly 30°C water to remove traces of acetone.
  5. Redistilled light petroleum (b.p.40-60°C) was then added to the ether followed by approx. 10% the volume of the organic layer of methanol/ 0.01 M-NH3 (4:1v/v).
  6. The solution was left to evaporate until it separated into clearly defined phases.
  7. The two phases were separated and the presence of pigments in only the bottom methanol phase was confirmed by fluorescence of solution under uv light.
  8. The sample was then collected and stored in methanol at -20°C.