User:Cheuk Ka Tong/Sandbox5/Protocols

=Cell-Free Systems=

Equipment

 * 37°C shaking incubator
 * 1L conical flasks x 3
 * Pipette fillers + pipettes (5ml, 10ml and 25ml)
 * Spectrometer + cuvettes
 * Weighing scale
 * Centrifuge + 150ml centrifuge tubes
 * Pipettes + pipette tips (20µl, 200µl and 1000µl)

Reagent

 * 2xYT medium
 * IPTG
 * Buffer A
 * 10mM Tris-acetate (pH 8.2)
 * 14mM Mg-acetate
 * 60mM K-lutamate
 * 1mM dithiothreitol (DTT)
 * 0.05% (v/v) 2-mercaptoethanol (2-ME)

Procedure
Growing the cells (Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)
 * 1) Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
 * 2) Add 1mM IPTG to cell culture to express T7 RNA polymerase.
 * 3) Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
 * 4) Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
 * 5) Centrifuge and weigh the wet cell pellets before storing them at -80°C.

Equipment

 * Pipette filler + pipettes (5ml, 10ml and 25ml)
 * Weighing scale
 * French press + French press cell
 * Centrifuge + 50ml centrifuge tubes
 * Pipette + pipette tips (20µl, 200µl, 1000µl)
 * 37°C shaking incubator
 * Dialysis membrane with molecular weight cut-off of 10,000
 * Magnetic stirrer
 * 4°C cold room

Reagent
(Note: For the ATP regenerating system in the pre-incubation solution, phosphoenolpyruvate and pyruvate kinase are used instead of creatine phosphate and creatine kinase. This is due to cost considerations.)
 * Buffer B
 * 10mM Tris-acetate (pH 8.2)
 * 14mM Mg-acetate
 * 60mM K-glutamate
 * 1mM DTT
 * Pre-incubation solution
 * 293.3mM Tris-acetate (pH 8.2)
 * 2mM Mg-acetate
 * 10.4mM ATP
 * 4.4mM DTT
 * 0.04mM amino acids
 * 16.9mM phosphoenolpyruvate
 * 0.77U/ml pyruvate kinase

Procedure
Lysing the cells
 * 1) Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
 * 2) Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.

Retaining the cell extract
 * 1) Centrifuge the crude lysate at 30,000RCF for 30min at 4°C.
 * 2) Carefully remove the top layer of the supernatant (lipid layer) and the pellet and centrifuge again.
 * 3) Shake the final supernatant at 100rpm.
 * 4) Gradually add 3ml of the pre-incubation solution to 10ml of the supernatant.
 * 5) Pre-incubate the supernatant with gentle shaking at 37°C for 80min. This degrades endogenous genetic content (DNA and mRNA).
 * 6) Dialyze the pre-incubated sample for 45min each at 4°C against 50 volumes of buffer B using a membrane with molecular weight cut-off of 10,000. Repeat the dialysis step three times.
 * 7) Centrifuge the retained extract at 4000RCF for 10min at 4°C to obtain the supernatant.
 * 8) Divide resulting S30 extract into small aliquots and store at -80°C.

(Note: Protease inhibitors are added to pre-incubation solution to prevent degradation of proteins required for gene expression.)

Equipment

 * 37°C shaking incubator
 * 1L conical flasks x 3
 * Pipette fillers + pipettes (5ml, 10ml and 25ml)
 * Spectrometer + cuvettes
 * Weighing scale
 * Centrifuge + 150ml centrifuge tubes
 * Pipettes + pipette tips (20µl, 200µl and 1000µl)

Reagent

 * 2xYT medium
 * IPTG
 * Buffer A
 * 10mM Tris-acetate (pH 8.2)
 * 14mM Mg-acetate
 * 60mM K-lutamate
 * 1mM dithiothreitol (DTT)
 * 0.05% (v/v) 2-mercaptoethanol (2-ME)

Procedure
Growing the cells (Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)
 * 1) Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
 * 2) Add 1mM IPTG to cell culture to express T7 RNA polymerase.
 * 3) Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
 * 4) Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
 * 5) Centrifuge and weigh the wet cell pellets before storing them at -80°C.

Equipment

 * Pipette filler + pipettes (5ml, 10ml and 25ml)
 * Weighing scale
 * French press + French press cell
 * Centrifuge + 50ml centrifuge tubes
 * Pipette + pipette tips (20µl, 200µl, 1000µl)
 * 37°C shaking incubator
 * Dialysis membrane with molecular weight cut-off of 10,000
 * Magnetic stirrer
 * 4°C cold room

Reagent

 * Buffer B
 * 10mM Tris-acetate (pH 8.2)
 * 14mM Mg-acetate
 * 60mM K-glutamate
 * 1mM DTT

Procedure
Lysing the cells
 * 1) Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
 * 2) Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.

Retaining the cell extract
 * 1) Centrifuge the crude lysate at 12,000RCF for 10min at 4°C.
 * 2) Carefully remove the top layer of the supernatant (lipid layer) and the pellet.
 * 3) Briefly pre-incubate the recovered supernatant at 37°C for 30min.
 * 4) Divide resulting S12 extract into small aliquots and store at -80°C.

Equipment

 * Nitrogen tap + plastic tubing
 * Desiccator connected to a vacuum
 * 100ml glass bottle
 * Sonicator with medium-sized probe
 * Ice bath
 * 25°C incubator
 * Pipette + pipette tips (1000µl)

Reagents

 * 10ml dodecane
 * 12.5µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%

Procedure
Preparing the lipid-oil suspension for the inner leaflet
 * 1) Place 125µl of the 20mg/ml DOPC solution in a 100ml glass bottle.
 * 2) With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
 * 3) Put the bottle in a desiccator connected to a vacuum for 1h.
 * 4) Add 50ml of mineral oil to reach a final lipid concentration of 0.05mg/ml.
 * 5) Set the sonicator probe to pulse 1, timer at 30mins.
 * 6) Put the bottle containing the suspension in the ice bath.
 * 7) Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating.
 * 8) Sonicate the suspension for 30mins.
 * 9) Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil.

Equipment

 * Magnetic stirrer
 * Centrifuge + 1-inch glass centrifuge tubes
 * Pipette + pipette tips (200µl, 1000µl)
 * 50ml glass tube
 * 5ml syringe
 * Long 16-gauge stainless steel needle

Reagents

 * 10ml ddH2O
 * Tris buffer
 * NaCl
 * Reporter

Procedure
Emulsifying the aqueous solution (while the interface settles)
 * 1) Separate about 5ml of the lipid-oil suspension into a glass container. This is for the interface preparation.
 * 2) Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
 * 3) Prepare solution B by adding a suitable quantity of reporter to 1ml of solution A.
 * 4) Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
 * 5) Gently stir the mixture with a magnetic stir bar for 3h.

Preparing the interface (to be done while the emulsion is mixing)
 * 1) Place 2ml of lipid-oil suspension over 3ml of solution A in a 1-inch-diameter centrifuge tube.
 * 2) Leave for 2–3h for lipids to achieve the coverage of the interface surface.

Forming the vesicles
 * 1) Pour 100µl of the inverted emulsion over the interface.
 * 2) Centrifuge at 120g for 10min.

Collecting the vesicles
 * 1) Using a 5ml syringe with a long 16-gauge stainless steel needle, collect some of solution A.
 * 2) Expel some of the solution to remove all air from the syringe and needle.
 * 3) With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe.
 * 4) Gently recirculate the buffer several times.
 * 5) Aspirate most of the solution into the syringe, and remove the needle from the solution.
 * 6) Wipe the tip of the needle clean.
 * 7) Unload the vesicle suspension into its final container.

(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)