IGEM:IMPERIAL/2007/Wet Lab/Protocols/CBD1.3

Aims:

• To solve the evaporation problem of the samples by either:
  • Preventing evaporation
  • Reducing evaporation drastically
  • Calibrate evaporation if it cannot be solved, so that the results will be able to account for that factor

Test 1:Test For How Oil Affects Fluorescence

We will test how a layer of oil will affect the fluorescence of a sample. We think that the oil may absorb some of the emitted photons and so fluorescence is expected to decrease. To test this we will made a series of GFP dilutions and measure how varying volumes of oil will affect the fluorescence measured. The GFP dilutions chosen had a similar range of fluorescence that is normally visualised within our cell free system. The control will be that we initially measure the GFP dilutions before addition of oil, and also we will keep one of each GFP concentration without oil. To limit the GFP that we use, we added oil serially so that small volumes were initially tested and to these more oil was added to increase the volume to then be tested. This relies upon quick measurements to limit the affect of GFP degradation.

Equipment

• Pipettes P5,P20,P200
• 96 Well Plate (black)
• Fluorometer
• Incubator 37°C

Reagents

• Distilled Water x 1200ul
• Parafin Oil x 270ul
• Mineral Oil x 270ul

Protocol

Prepare GFP Dilutions
We use a sample of GFP of unknown concentration to prepare a series of dilutions. We initially tested several dilutions to determine a range of 3 suitable concentrations to cover the low, medium and high fluorescence signals that we see in our experiments. The dilutions used depend on the initial GFP sample and so it is better to do some rough dilutions before deciding up final dilutions. The dilutions we used were as following:

• 1ul GFP + 59ul Distilled Water
• 5ul GFP + 55ul Distilled Water
• 20ul GFP + 40ul Distilled Water

Load Plate

• Add the following dilutions of GFP to the following wells following the schematic.
• Take an inital reading of the GFP dilutions

10ul of Oil

• Then add 10ul of Paraffin and Mineral Oil to the to the following wells following the schematic. A p20 should be used to measure out the oil, in addition when the oil is added to the well it should be carefully dropped into the centre if the well
• Take an inital reading of the GFP dilutions

20ul of oil

• Add 10ul more of Paraffin and Mineral Oil to the to the following wells following the schematic, this is to bring the oil volume to 20ul. A p20 should be used to measure out the oil, in addition when the oil is added to the well it should be carefully dropped into the centre if the well
• Take an inital reading of the GFP dilutions

90ul of oil

• Add 70ul more of Paraffin and Mineral Oil to the to the following wells following the schematic, this is to bring the oil volume to 90ul. A p200 should be used to measure out the oil, in addition when the oil is added to the well it should be carefully dropped into the centre if the well
• Take an inital reading of the GFP dilutions

Test 2: Testing Evaporation when using Oil

We have decided to test how we can prevent evaporation using oragnic oils. Two types of oil will be tested, parafin oil and mineral oil, at varying volumes to see which is a suitable volume of oil to use. We will be testing 60ul of water with varying volumes of mineral oil and parafin oil. The samples will then be left overnight and measured again in the morning to determine the rate of evaporation. This rate will then be compared to the control which is just water with no oil.

Reagents

• Distilled Water x 1200ul
• Parafin Oil x 270ul
• Mineral Oil x 270ul

Equipment

• Pipettes P5,P20,P200
• 96 Well Plate (black)
• Fluorometer
• Incubator 37°C

Protocol

Day 1
Following the following schemetic first pipette 60ul of water into each well. Then pipette in the required volume and type of oil Note that oil can be tricky to pipette because of the capillary action of pipettes, be aware of this. Place the plate in the incubator at 37^oC

Day 2
To measure the evaporation we use a gilson pipette. To do this adjust the volume to a suitable volume and remove the sample until all is removed. Note down the volume removed and minus this from the original total volume. When we carried this out we started at 20ul and then adjusted down to 5ul when 20ul was no longer appropriate. It is best to judge the measurement based on the total sample volume and adjust accordingly. Remember that in a well samples can get stuck to the rim of the well.

Well	Water Added(ul)	Paraffin Oil	Total Volume (ul)
E5	60ul	10ul	70ul
F5	60ul	10ul	70ul
G5	60ul	10ul	70ul
E6	60ul	20ul	80ul
F6	60ul	20ul	80ul
G6	60ul	20ul	80ul
E7	60ul	90ul	150ul
F7	60ul	90ul	150ul
G7	60ul	90ul	150ul
B5	60ul	10ul	70ul
C5	60ul	10ul	70ul
D5	60ul	10ul	70ul
B6	60ul	20ul	80ul
C6	60ul	20ul	80ul
D6	60ul	20ul	80ul
B7	60ul	90ul	150ul
C7	60ul	90ul	150ul
D7	60ul	90ul	150ul

Test 3: 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