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

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(Construction of pT7-GFP)
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*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
*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
*Measurements will carry on over tomorrow day to give ~30hours of measurements
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==Midiprep of Biobricks==
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#Midipreped sample left overnight in isopropanol was washed with 70% ethanol <br>Previous protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Midiprep|Midiprep]] in the general protocols page
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#Solution was dissolved in 300 &micro;l of ddH<sub>2</sub>O
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#Resulting concentration of DNA was 40 ng/&micro;l (lower than expected!)

Revision as of 07:54, 3 September 2007

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Contents

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!)
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