IGEM:IMPERIAL/2007/Projects/Experimental Design/Improve Methodology/Protocol3

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Testing the expression of the samples when using Oil

After finding out the type and amount of oil to use on our samples, through the previous tests, we will test how the layer of oil affects the oxygen supply to the DNA constructs. This will be done by measuring the change in fluorescence of a sample, with the oil layer, over time and comparing it with positive and negative controls. We will be testing 40ul of cell extract, with 20ul pTet DNA construct and 30ul of paraffin oil. Our negative control will consist of 40ul of cell extract, with 20ul control DNA and 30ul of paraffin oil. The positive control will consist of 40ul of cell extract and 20ul of pTet DNA. The samples will be measured every 30mins over a period of 5hours. As readings can’t be taken overnight, the samples may be left overnight and measurements can be carried on again in the morning.

Reagents

  • Distilled Water
  • Paraffin Oil
  • pTet DNA
  • Control DNA empty vector
  • S30 cell extract (with premix solution and amino acids)

Equipment

  • Pipettes P5,P20,P200
  • Plate
  • Fluorometer

Protocol

Day 1:
Following the schematic given below pipette 40ul of cell extract mixture into each well. Then put 20ul of the right DNA in to the wells as shown below. Before putting the oil, take an initial reading of the samples. Then pipette in 30ul of paraffin oil into the right wells. As the oil is very tricky to pipette, use a P20 pipette instead of a P200 to put the Paraffin oil into the wells (2 x 15ul). Pipetting slowly ensures the right amount is pipetted up and down.

Take a fluorometer reading immediately after pipetting the oil into the wells. Incubate the plate at 37oC. Take a reading every 30minutes after this.

Day 2:
Carry on taking a reading every 30mins. After taking the last reading, measure the volume of samples left in the wells to ensure no evaporation has occurred.

Well Test Construct In vitro chassis
D5 40ul CE + 20ul pTet 30ul of Paraffin oil
D7 40ul CE + 20ul pTet 30ul of Paraffin oil
E6 40ul CE + 20ul control DNA -
E9 40ul CE + 20ul pTet 30ul of Paraffin oil
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