Team:Warsaw/Calendar-Main/30 September 2009

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Bistable switch testing

Monika

Tasks:

  • Transformation of chemocompetent cells (Top10) with plasmid containing bistable switch


Methods:

  • Transformation of chemocompetent cells with 10μl of DNA solution - procedure destribed here
  • Planting on LB-agar medium supplemented with tetacycline


Results: Successful, many colonies that express GFP.


Cloning of AraC coding sequence

Justyna


Task 1:

Overnight digestion in 37°C, volume of 30µl

Comment:

Enzymes:
BBa_B0032 part: SpeI + PstI
BBa_C0080 part: XbaI + PstI


Results:




Task 2:

  • Ligation of AraC gene and BBa_B0032

4 hour long ligation in room temperature, volume of 25µl

Ligation mix:

BBa_B0032 7µl (~49ng)
AraC sequence  15.5µl (~70ng)
buffer 2.5µl
ligase 1µl (Fermentas)

Fusion of YFP coding sequence and TetR coding sequence

Justyna


Task 1:

Overnight digestion in 37°C, volume of 30µl

Comment:

Enzymes:
BBa_C0040+BBa_R0080 part: SpeI + PstI
BBa_E0032 part: XbaI + PstI


Results:

above.


Task 2:

  • Ligation of YFP gene and BBa_C0040+BBa_R0080

4 hour long ligation in room temperature, volume of 20µl

Ligation mix:

BBa_C0040+BBa_R0080 7µl (~50ng)
YFP sequence  9.5µl (~114ng)
buffer 2.0µl
ligase 1µl (Fermentas)

April
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