Difference between revisions of "Team:Macquarie Australia/Experiments"

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 18-22^oC, 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 4^oC

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 4^oC, 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 -80^oC

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 @37^oC, then 20mins @80^oC).

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 37^oC for 60 mins, followed by a deactivation step at 80^oC 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 37^oC 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 37^oC 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 4^oC (on ice).

17. Digest overnight at 37^oC.

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 4^oC 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

○