IGEM:IMPERIAL/2007/Projects/Experimental Design/Improve Methodology

Problems with current methodology

The data we got from our experiments didn't seem to fit in with what we expected from our models and prior knowledge. We have come up with a few considerations for our experimental design that might give us better results from our experiments

Problems

1. Evaporation - is a major problem as:
   • changes the flourescence of the samples
   • changes the context
2. Mixing of the samples - was not done regularly or constantly (i.e. was done manually), which:
   • affects reactions - disruptions the reaction mixture
   • affects the fluorescence
3. Temperature regualtion - the temperature of the sample kept varying due to the sampling:
   • about 5mins to get one reading - sampling time of 10mins means sample not in a steady temperature - therefore need a bigger sampling time or reduce time to get readings

Solutions

1. Mineral oil - to reduce evaporation of samples. Need to test as:
   • Might affect the reading of the fluorometer, can probably be calibrated?
   • May block the oxygen needed by the reaction mixture
   • Need to get optimum volume of mineral oil which minimises evaporation whhile not blocking all oxygen and allowing fluoromemter readings
2. Double the volume of sample - would mean that less of the sample is evaporated in proportion to the total volume, but:
   • doesn't minimise the evaporation
3. PCR - put the samples in PCR tubes - the heated lid would prevent condensation, but:
   • would need to keep pipetting the samples from the PCR machine to the fluorometer and back for the readings - big problem for 24hr experiments
4. Humidity - increase the humidity around the samples to reduce evaporation
   • Fill the wells surrounding the wells, with the samples, with water

Tests

1. Test for difference in fluorescence and the volume of samples wrt mineral oil being added
   • GFP vs GFP + mineral oil/Paraffin oil
   • Protocol
   • Results
2. Test for rate of evaporation wrt volume of mineral oil being used
   • H₂O vs H₂O + mineral oil (different volumes)
   • Protocol
   • Results
3. Test for chromophore formation - ie test if oxygen still reaching the sample
   • Cell extract + DNA vs cell extract + DNA + Mineral oil
   • Protocol
   • Results

More problems

1. Evaporation still occured in the samples with 30µl paraffin oil, over a 21hour period (Test 3 above)
2. Possible reasons:
   • Oil volume not enough to cover all of the surface of the samples
   • Shaking might have disrupted the oil - causing it to form big droplets, leaving sample uncovered
     • but no shaking was done overnight, so the oil layer should have been able to form again
   • Paraffin oil is denser than cell extract mixture so it forms a layer at the bottom
     • Did a test today which proves paraffin (and mineral) oil less denser than cell extract
   • Some contents of the cell extract could have dissolved in the oil

Way forward

1. Try the humidity method (described above):
   • Test whether having water samples surrounding the actual samples decreases the evaporation or not
2. Create a calibration curve for evaporation over time:
   • experiment to find out how the volume change over time - use the change in mass instead of pipetting each time to get volume