Revision as of 00:32, 16 October 2018

Cloning

Linearisation pETDuet1 and pRSFDuet1 plasmid backbones using PCR

Forward and reverse primers created for the plasmids
2 uL of plasmid + 198 uL of water

Component			50 µl Reaction	Final Concentration
Q5 High-Fidelity 2X Master Mix			25 µl	1X
10 µM Forward Primer			2.5 µl	0.5 µM
10 µM Reverse Primer			2.5 µl	0.5 µM
Template DNA			2 uL DILUTED	< 1,000 ng
Nuclease-Free Water			18 uL
STEP	TEMP		TIME
Initial Denaturation	98°C		30 seconds
25–35 Cycles	98°C		5–10 seconds
	62		10–30 seconds
	72°C		2 min
Final Extension	72°C		2 minutes
Hold		4–10°C

Plasmid digest (Dpn1 digest)

Set-up the reaction mixture: Code:

Restriction Enzyme

1µl

DNA

1 µg

10X Cutsmart

5 µl (1X)

Total Reaction Volume

50 µl
Incubate for 1 hr at 37C
Heat inactivate at 80C for 20 minutes

Agarose gel electrophoresis

Combine 100x agarose powder with 1x TAE buffer in a microwavable flask (eg. 1 g of agarose for 100 mL of TAE). The volume of agarose gel will depend on the size of gel you are making.
Microwave for 1-2 min until the agarose is completely dissolved (do not overboil the solution). Stop and swirl the flask every 20 seconds and until the solution is as clear as water.
Let agarose solution cool down to about 50 C (when you can comfortably hold the flask with your hand), then add 1 uL of RedSafe to the agarose solution.
Seal the ends of a gel tray using masking tape. Pour the agarose into the gel tray with a well comb in place. Let the gel sit at room temperature for 20-30 mins until the gel solidifies.
Place the gel into the gel box, fill the gel box with 1 x TAE buffer until the gel is covered then remove the well comb.
Mix 2 uL of the digest sample with 3 uL of H2O and 1 uL of 6x loading dye.
Load 5 uL of 1kb DNA ladder into the first lane of the gel. Load the remaining digest samples into the gel. Remember to include a negative control (non-digested plasmid).
Connect the gel box to a power pack and run the gel at 100V for 1 hr. You should be able to see small bubbles rising in the buffer solution immediately after you turn the power pack on.
Carefully take the gel tray to the spectrophotometer and analyse the DNA fragments with UV light. We expect to see a single clear band in digested samples, and a smear for the undigested plasmid at a higher position. Smear and a clear band indicates incomplete digestion.

Gibson Assembly

Materials:

5X Isothermal Reaction Mix (6 mL total)
- 3 mL 1 M Tris-Hcl (pH 7.5)
- 300 μL 1 M MgCl2
- 60 μL 100 mM dGTP
- 60 μL 100 mM dATP
- 60 μL 100 mM dTTP
- 60 μL 100 mM dCTP
- 300 μL 1 M DTT
- 1.5 g PEG-8000
- 300 μL 100 mM NAD
- 360 µL water
- Store at -20°C.
Assembly Master Mix (1.2 mL total)
- 320 μL 5X Isothermal Master Mix
- 0.64 μL 10 U/μL T5 exonuclease
- 20 μL 2 U/μL Phusion DNA Pol
- 0.16 μL 40 U/μL T4 DNA Ligase
- 860 μL water
- Store in 15 µL aliquots at -20°C.

PCR or digest your fragment of choice and gel purify
If PCR from a methylated DNA template (e.g. propagated plasmid), a DpnI digest can be used to remove the unwanted plasmid. Clean up afterwards.
Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
Add 5 μl of DNA to be assembled to the master mixture.
The DNA fragments should be in equimolar amounts.
Small fragments (<1 kb) must be added in a five times excess
You can calculate the quantity of each fragment using their molecular weights.
Alternatively, you can use the length of each fragment as a proxy for the molecular weight (assuming similar GC content in all fragments).
Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
Incubate at 50 °C for 15 to 60 min (60 min is optimal).

Heat shock transformation

Incubate 50ng of plasmid construct with 25 µL of chemically competent E. coli T7
Express cells or E. coli DH5α on ice for 30 minutes.
Heat shock the cells for 45 seconds at 42C and placed back onto ice for 2 minutes.
Allow cells to grow for 45 minutes in 200 µL of SOC outgrowth media (NEB) at 37°C and 200 rpm.
Spread plate onto Luria broth (LB) agar plates containing 100 µg/mL of ampicillin and grown at 37°C overnight.

Colony PCR

Pick up 5 individual bacterial colonies from each plate that was grown overnight using a pipette tip and dilute each colony into 50 uL of water. Label A, B, C, D, E etc.

Create the colony PCR master mix. Determine volumes for the cPCR master mix by multiplying (the number of reactions + 2) by each volume below:

18 uL Nuclease free water
5 uL 5x Taq master mix
0.5 uL 10 uM T7 promoter primer
0.5 uL 10 uM T7 terminator primer

Example, for 20 colonies, you would add 22 x 18 uL of water, 22 x 5 uL of 5x Taq master mix, 22 x 0.5 uL of forward primer and 22 x 0.5 uL of reverse primer.

Add 1 uL of each bacterial dilution and 24 uL of cPCR master mix to a PCR tube.

Run PCR with the following instructions (lid at 105 C and volume = 20 uL):

95 C 5:00 mins
95 C 0:30 mins
55 C 0:30 mins
68 C 2:00 mins
68 C 5:00 mins
4 C 0:00 mins (hold)

Run PCR products on a 1% agarose gel, TAE running buffer, 100V, 1 hr. 10 uL of PCR product + 2 uL of 6x loading dye, and have 100bp ladder in the first lane.

Sequencing

Transfer 10 uL of purified plasmid sample (50-100ng/uL) to an Eppendorf tube. Add 5 uL of one primer.
Request sequencing. http://sequencing.ramaciotti.unsw.edu.au/index.html
Label Eppendorf tubes with the order number, and 1, 2, 3, etc.
Take Eppendorf tubes to the new Biosciences building Lvl 2 (at UNSW, Sydney, Australia), and store the samples in the fridge provided.
Sanger sequencing is carried out following the provided protocol.

Restriction cloning

Set-up the reaction

Restriction Enzyme

1µl of each enzyme

DNA

1 µg

10X Cutsmart

5 µl (1X)

Total Reaction Volume

50 µl
Incubate for 1 hr at 37C
Heat inactivate at 80C for 20 minutes

Ligation

Set up the following reaction in a microcentrifuge tube on ice:

2 μl

Component	20 μl Reaction
T4 DNA Ligase Buffer (10X)*
Vector DNA	50 ng
Insert DNA	A molar ratio of 1:3 vector to insert should be used
Nuclease-free water	to 20 μl
T4 DNA Ligase	1 μl

Gently mix the reaction by pipetting up and down and microfuge briefly.

For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

Heat inactivate at 65°C for 10 minutes.

Chill on ice and transform 1-5 μl of the reaction into 25 μl competent cells.

Miniprep

Protocols were followed from the Qiagen QIAprep Spin Miniprep Kit. No changes were made.

Protein Expression and Purification

Starter culture

One colony was selected from the plate grown overnight and grown in 2 mL of LB containing 2 uL of the appropriate antibiotic at 37°C and 200 rpm and left overnight.

Large-scale grow-up

Baffled shake flasks containing 500 mL of LB with 50uL of the appropriate antibiotic at 37°C are inoculated with the starter culture.
The cells are grown at 37°C and 200 rpm and OD600 is periodically measured.
Once OD600 reaches above 0.6, add IPTG of 1 mM concentration to induce the expression the proteins.
After induction, grow the cells overnight at 24°C, 200 rpm.

Collection of cells by centrifugation

Centrifuge the bacterial culture at 4600 x g for 20 minutes.
Collect cell pellet and resuspended in binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).

Cell lysis by sonication

Lyse the cell pellet by sonication (Branson) for 10 minutes at 50% amplitude at alternating 2 second intervals, kept on ice.
Centrifuge the cell lysate at 15000 rpm for 45 minutes.
Collect the supernatant (soluble fraction).

IMAC

(Immobilised metal ion affinity chromatography (IMAC) was performed to purify the expressed proteins.)

His-tagged protein is bound to a 1 mL Ni-NTA Superflow Cartridge (Qiagen) by loading the soluble fraction of the cell lysate onto the column.
Wash with 10 mL of binding buffer (20mm NaH2PO4, 500mM NaCl, 10mM Imidazole).
Elute with 2 mL of elution buffer (same as binding, but with 500 mM imidazole).
Collect fractions with SDS-PAGE.

Buffer exchange

Column

Elutions were analysed with SDS-PAGE and buffer exchanged into PBS pH 8 using Pierce Protein Concentrators PES, 10K MWCO, 2-6 mL (Thermo Scientific).
Add protein to the column
Top up column with PBS buffer
Centrifuge column at 4600 x g for 20 minutes.
Repeatedly centrifuge, discard flow through, and top up with PBS buffer (pH 8) until dilution factor of 0.1 is reached. That is, there is 1% of the old buffer left in the solution.

Dialysis

Add 1mL of protein and 1mL of PBS buffer (PH 8) to a 15mL Falcon tube.
Add 2mL of the solution to a SnakeSkin™ Dialysis Tubing, 10K MWCO, 22 mm.
Use dialysis tubing clamps (one-piece polypropylene clamp) to further secure the solution inside the snakeskin dialysis tubing.
Add 500mL of PBS buffer, pH 8, (this is the buffer we want to exchange into) into a 500ml glass beaker.
Place the dialysis tubing with the solution into the beaker.
Place the beaker on top of a magnetic stirrer, 75 rpm, and leave overnight.

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