IGEM:IMPERIAL/2007/Notebook/2007-8-30

<calendar> name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

Construction of pT7-GFP

1. Insert re-digest was run on 1% agarose gel
   • 1. 30 μl insert re-digest
   • 2. 5 μl uncut insert
   • 3. 2 μl 1 kb DNA ladder

Protocols can be found at Electrophoresis in the general protocols page

1. PCR purified vector digest
2. Gel extract purified insert re-digest

Protocols can be found at DNA Extaction/Purification in the general protocols page

1. Purified vector and insert were run on 1% agarose gel
   • 1. 6 μl purified insert
   • 2. 3 μl purified vector
   • 3. 2 μl 1 kb DNA ladder

Protocols can be found at Electrophoresis in the general protocols page

1. Ligated vector and insert at 14°C overnight
   • 7 μl purified insert
   • 1 μl purified vector
   • 1 μl T4 ligase
   • 1 μl T4 ligation buffer
2. A negative control with ddH20 instead of insert was also set up

Cell by date - Operating Temperature Range

Construct - pTet-GFP BBa_I13522
Temperatures - 37 °C and 20°C Aims: To test for the behaviour of the DNA construct (pTet) at temperatures 20oC and 37oC by observing the amount of fluorescence produced over a period of 24 hours.

• For each temperature two experiments carried out at staggered time points to minimize the time points not measured over night.
• Sampling was initially every 10 minutes however, this was reaccessed and changed to 20 and 30 minute time intervals.
• Measurements will carry on over tomorrow day to give ~30hours of measurements.

Protocol can be found here under Phase 2-Operating Temperature Range on the experimental design page.

Results can here under Results on the experimental design page.

Degradation of GFP

• Tested GFP degradation at 37 °C and 20°C
• The tests were carried out on the same plate as the Operating Temperature Ranges and so the sampling was the same as that for the individual temperatures
• Measurements will carry on over tomorrow day to give ~30hours of measurements

Midiprep of Biobricks

1. Midipreped sample left overnight in isopropanol was washed with 70% ethanol
   Previous protocols can be found at Midiprep in the general protocols page
2. Solution was dissolved in 300 µl of ddH₂O
3. Resulting concentration of DNA was 40 ng/µl (lower than expected!)