Beruflich Dokumente
Kultur Dokumente
Objective
To create Chlorogel with the addition of buffered chloroplast stock solution into an ethanol-free silica
sol-gel prepared by rotavaporisation.
Equipment
i) Buffer preparation
- Anhydrous Na2HPO4
- Anhydrous KH2PO4
- Anhydrous KCl
- Sucrose sugar
- Distilled water
- Aqueous KOH or HCl (any conc.)
- 1 L glass beaker
- Glass stirrer
- Spoon
- pH meter
- Thermometer
- Parafilm
ii) Chloroplast isolation
- Fresh spinach leafs
- Distilled water
- Blender
- Cold sucrose-phosphate buffer
- Bandages
- Centrifuge tubes 50 mL
- Centrifuge
- Micropipette
- Glass funnel
- Measuring cylinder 50 mL
- Lamp/light source
- Ice bucket w/ ice
iii) Sol-gel process
- Aqueous tetraethyl orthosilicate (TEOS)
- Aqueous 0.05 M HCl
- Distilled water
- Evaporating flask
- Beaker w/ ice
- Magnetic stirrer
- Measuring cylinder 25 mL
- Ultrasonic processor
- Retort stand + clamp
- Thermometer
- Stopwatch
iv) Rotavaporisation process
- Rotavapor unit
- Vaseline
- Stopwatch
- Glass stirrer
- Container mold
Procedure
i) Buffer preparation (Estimated completion time: 20 min)
- Add 3.97 g of Na2HPO4 in 700 mL of distilled water and stir to completion.
- Add 2.99 g of KH2PO4 into solution and stir to completion.
- Add 136.9 g of sucrose and stir to completion.
- Add 0.745 g of KCl and stir to completion.
- Add distilled water to give a volume of 950 mL.
- Adjust pH of solution with KOH or HCl to 7.3.
- Add distilled water to give a final volume of 1 L.
- Sucrose-phosphate buffer hence obtained; stored at 4°C.
ii) Chloroplast isolation (Estimated completion time: 40 min)
- Wash approximately 50 g of fresh deveined spinach leafs with distilled water in the
presence of a light source for 10 minutes.
- Homogenize spinach leafs in blender with the addition of 200mL sucrose-phosphate
buffer for 10 s.
- Filter homogenate through 3 layers of bandages.
- Equally decant filtrate into an even number of (boiling-tube size) centrifuge tubes.
- Centrifuge at 1500x for 10 min.
- Discard supernatant and resuspend the pellets in 35 mL of sucrose-phosphate buffer.
- Centrifuge at 1500x for 10 min.
- Discard supernatant and resuspend the pellets in 15 mL of sucrose-phosphate buffer.
- Stock chloroplast solution hence obtained; stored at 4°C in subdued light.
iii) Sol-gel process (Estimated completion time: 60 min)
- Prepare an evaporating flask on beaker filled with cool ice at 4°C.
- Add 11.0mL of TEOS, 4.0 mL of distilled water and 1.55 mL of 0.05M HCl.
- Stir mixture vigorously for 30 min.
- Subject vessel onto an ultrasonic processor at 20-kHz freqeuency and 2.4 kJ/cm3 ultrasonic
energy for 15 min.
- Homogenous sol hence obtained.
iv) Rotavaporisation process (Estimated completion time: 35 min)
- Fix evaporating flask into the rotavapor.
- Commence process at 42°C for 25 min of controlled vacuum at 20 bar.
- Quickly decant mixture from evaporating flask into container mold and stir with stock
chloroplast solution.
- Leave sol-gel to cure for ? hours.
- Chlorogel hence obtained.
Precaution / Remarks
i) Buffer preparation
- Care must be taken not to contaminate the anhydrous salts. Always wash spoon with
distilled water and wipe with cloth each time after use.
- Crush solute into grains to ensure accurate weighing and better solubility.
- Always keep the two electrodes of the pH meter in uncontaminated distilled water.
- Take extra care not to add too much KOH or HCl into the buffer when adjusting pH
- Once buffer is prepared, cover beaker surface with parafilm to prevent contamination.
- Must be refrigerated to 4°C or less prior to chloroplast isolation experiment.
- Sucrose is used to regulate osmotic pressure of the buffer around the chloroplasts,
whilst Na2HPO4 and KH2PO4 are a good source of Na+ and K+ ions for regulating ion
concentration. H+ and PO2-4 ions maintain the proton gradient and supply inorganic phosphate
ions for ATP synthesis in chloroplasts respectively.
ii) Chloroplast isolation
- Blender, centrifuge tubes, measuring cylinders and any other containing vessels used
to house the chloroplast mixture must be pre-cooled to 4°C or less prior to the experiment.
Sterilization is necessary to prevent microbial growth in the vessels.
- Experiment must be swiftly conducted to ensure survival of chloroplasts from
changes in the external environment.
- Fresh, healthy and locally-grown spinaches harvested after 3-4 weeks must be used.
- Washing in the presence of light necessary to maintain chloroplast activity and
remove unwanted stuffs from spinach leaves.
- Stop blending after 10 seconds has gone, or once the mixture looks homogenized.
- An even number of similarly-weighted centrifuge tubes must be oppositely placed to
each other in order to balance the centrifuge rotor.
- Parafilm to be used to seal the vessels containing stock chloroplast solution to
prevent microbial contamination.
- Stock chloroplast solution to be kept at 4°C to preserve chloroplast survivability.
- Homogenization of deveined spinach leaves and filtration separates the plant cells
from the leaves. Centrifugation is performed twice; the first to separate plant cells from the
homogenized mixture, the second to separate chloroplasts from plant cells. Resuspension in
phosphate buffer solution necessary to retain chloroplast activity. Optionally, a density
gradient centrifugation can be subsequently performed to separate the intact chloroplasts
from broken chloroplasts.
iii) Sol-gel process
- Tetraethyl orthosilicate, ethyl silicate, silicic acid, tetraethyl ester, silicon ethoxide or TEOS is
labelled as Xn, a harmful hazard. Its smell is akin to that of ester. Sniffing and consumption is
not advised. Rinse with water if in contact with body.
- Add reagents in the specified volumes, otherwise the sol-gel process will be affected.
- The whole evaporating flask must be completely immersed in ice-filled beaker and wrapped
with aluminium foil to prevent drastic temperature change and melting.
- Clamp evaporating flask to a retort stand and immerse contents in the ultrasonic processor.
The water level must exceed the sol-gel level inside the evaporating flask to ensure its
contents are subjected to ultrasonic waves.
- Stirring and exposure to ultrasonic waves assist in the hydrolysis of TEOS and the
condensation of two silanol groups 2(Si-OH) into one siloxane bridge (Si-O-Si).
iv) Rotavaporisation process
- Ensure that the evaporating flask is firmly attached to the receiver with a clamp.
- Lubricate the receiver with Vaseline to ensure smooth rotation.
- Entire evaporating flask must be immersed in bath before distillation.
- Wear gloves to prevent scalding when handling evaporating flasks.
- At the end of the distillation, immediately detach evaporating flask and transfer contents into a
container mold. Wash vessel thoroughly with distilled water to prevent formation of glass.
- Stir the contents of the container mold to uniformly distribute chloroplasts within the media.