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

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

Construction of pT7-GFP

 * 1) Insert re-digest was run on 1% agarose gel
 * 2) *1. 30 μl insert re-digest
 * 3) *2. 5 μl uncut insert
 * 4) *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
 * 2) *1. 6 μl purified insert
 * 3) *2. 3 μl purified vector
 * 4) *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
 * 2) *7 μl purified insert
 * 3) *1 μl purified vector
 * 4) *1 μl T4 ligase
 * 5) *1 μl T4 ligation buffer
 * 6) 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 &deg;C and 20&deg;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 &deg;C and 20&deg;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 &micro;l of ddH2O
 * 3) Resulting concentration of DNA was 40 ng/&micro;l (lower than expected!)

Vesicles
Results Samples 1 through 5, prepared the day before were collected for observation under the microscope. The following results were obtained:


 * Vesicles were found in sample 1.
 * A single vesicle was found in sample 2.
 * No vesicles were found in samples 3-5, in spite of the presence of many GFP aggregates.