Difference between revisions of "Team:Oxford/Test/Beads"

Aim

To improve on last years Oxford iGEM team biobead design, using agarose to make up beads and then coating them with a ethyl acetate and polystyrene mixture.

Method

In a fume cupboard, break up a standard (polystyrene) petri dish into small pieces and dissolve in the minimum amount of ethyl acetate.

Prepare a 150 mL 1.5 % Agarose solution in breaker with screw cap. Microwave on high for 2 minutes and then cool to 40 °C in water bath, when microwaving ensure the screw cap is placed on loosely.

Remove agarose solution from water bath and bring to laminar airflow cupboard. Pour the agarose into petri dish to a depth of 1cm. Allow to set, this should take roughly 15 minutes.

Still under laminar flow, use an autoclaved 1 cm diameter hole borer to core out the required number of identical agarose cylinders and place in a second petri dish and allow drying.

Bring uncoated ‘beads’ to fume cupboard. Using autoclaved needles, pick up individual beads and dip in ethyl acetate-polystyrene solution, stand each bead on needle upright in fume cupboard on blob of blu-tac to allow ethyl acetate evaporation and the coating to set. When the coating is almost set the beads should be able to be handled through gloves without damaging the coat. Remove needle and mould the polymer coat over the needle hole, sealing them.

Control of substances Hazardous to Health (COSHH) Assessment

Agarose

• Not a hazardous substance

Ethyl Acetate

• H225 – Highly flammable liquid and vapour.
• H319 – Causes serious eye irritation.
• H336 – May cause drowsiness or dizziness.>
• P210 – Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking.
• P261 – Avoid breathing vapours.
• P305+P351+P338 – IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do so. Continue rinsing.

Ethyl acetate poses the biggest safety hazard in this experiment so therefore do all steps involving it in the fumehood. Be careful of cutting self when breaking up the Petri dish, as the pieces can be sharp.

Write Up

150mL of 1.5% agarose was made up using 2.25g of agarose powder and 150 mL of MilliQ.

In a fumehood, one standard petri dish was broken into small pieces and placed into a 500mL beaker. To this, 50 mL of ethyl acetate was added along with a stirrer bar and the mixture was left to stir for 10 minutes. After 10 minutes the petri dish was still not dissolved so a further 10 mL of ethyl acetate was added and left to stir again.

The agarose was then poured into a petri dish to a depth of 1cm and left to set for 15 minutes. After this was set, using an eppendorf tube with the lid cut off cylinders of agarose were cut out and placed into a second petri dish.

After the agarose was left to dry it was taken to the fumehood, to be coated. The agarose beads were placed onto the end of pipette tips and then dipped into the ethyl acetate-polystyrene mixture. These were then left to dry standing up on the pipette tips.

The beads were left to dry overnight and it was found that the coating did not stick sufficiently to the agarose.

Findings

• Beads are far too big to fit into a catheter, but could be used in larger pipes.
• It is very hard to get an even coating of the ethyl acetate-polystyrene mixture around the bead and it would tend to slide off.
• Ethyl acetate-Polystyrene mixture was difficult to clean up.
• Is the coating actually porous?

Aim

Try and make some beads out of alginate instead of agarose and see if we can make them smaller and more suitable to be placed inside a catheter.

Method

Make the 30ml of 1.2% alginate solution in dH2O with 0.9% NaCl, add 0.36g of alginate slowly to hot water followed by 0.27g of NaCl and add a magnetic stirrer bar. Then leave to stir until all solid is dissolved.

To compare, make up 30ml of 1.2% alginate solution in dH2O. Add 0.36g of alginate slowly to hot water and add a magnetic stirrer bar. Then leave to stir all solid is dissolved.

Transfer this mixture in a 2mL syringe with a needle attached and drop the mixture into a solution of 0.1M calcium chloride. Vary the size of the syringe and height from which you drop and record the effect on the beads. Leave the beads in the CaCl2 solution for 5 minutes and then filter the beads out.

Control of substances Hazardous to Health (COSHH) Assessment

Sodium Aliginate

• Not a hazardous substance

Sodium Chloride

• Not a hazardous substance

Calcium Chloride Solution (0.1M)

• H319 – Causes serious eye irritation
• P305 + P351 + P338 - IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

The calcium chloride poses safety hazards therefore when doing steps involving make sure you are wearing safety goggles and gloves.