IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/13

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Raphaël

Restriction digest 2h, 37°C (10µL of minipreped DNA)

  • BBa_J23119 cut by SpeI + PstI
  • BBa_J23110 cut by SpeI + PstI
  • BBa_J37033 cut by XbaI + PstI
  • BBa_R0062 cut by SpeI + PstI
  • BBa_E0240 cut by XbaI + PstI


Raphaël and Léa

DNA Gel electrophoresis (agarose 1,0% w/v) (gel extraction for purification of DNA)
After migration, we made a bath with EtBr during 5-10 min.

Protocol (6 wells)

  • 10 µL of ladder 1Kb
  • 17 µL of digested DNA (J23119 - SpeI+PstI) + 3 µL of loading buffer 6x
  • 17 µL of digested DNA (J23110 - SpeI+PstI) + 3 µL of loading buffer 6x
  • 17 µL of digested DNA (R0062 - SpeI+PstI) + 3 µL of loading buffer 6x
  • 17 µL of digested DNA (E0240 - XbaI+PstI) + 3 µL of loading buffer 6x
  • 17 µL of digested DNA (J37033 - XbaI+PstI) + 3 µL of loading buffer 6x

Migration : 50V, 1h30
Image:extractionbefore_biobrick_130710.jpg Image:extractionafter_biobrick_130710.jpg
double digestion of J37033 (LuxR) failed.

Gel extraction - Kit Qiagen

Ligation (T4 ligase), 1h at room temperature
vector : R0062 (Plux) cut by SpeI + PstI
insert : E0240 (RBS+GFP+ter) cut by XbaI + PstI

Transformation
Transformation into TOP10 - pSB AmpR with biobricks - Plating 100µL of each on the plates (Amp) - Incubation overnight :

  • Product of ligation (R0062+E0240)

-> Transformation didn't work

Restriction digest 1h, 37°C (10µL of minipreped DNA)
Again because the double digestion of J37033 didn't work and we have forgotten to digest 2 other biobricks

  • BBa_J23119 cut by SpeI + PstI
  • BBa_J23110 cut by SpeI + PstI
  • BBa_J37033 cut by XbaI + PstI
  • BBa_R0062 cut by SpeI + PstI
  • BBa_E0240 cut by XbaI + PstI
  • BBa_K081008 cut by SpeI + EcoRI
  • BBa_B0015 cut by EcoRI + XbaI

DNA Gel electrophoresis (agarose 1,0% w/v) (gel extraction for purification of DNA)
After migration, we made a bath with EtBr during 5-10 min.

Protocol (6 wells)

  • 10 µL of ladder 1Kb
  • 17 µL of digested DNA (J37033 - XbaI+PstI) + 3 µL of loading buffer 6x
  • 17 µL of digested DNA (B0015 - EcoRI+XbaI) + 3 µL of loading buffer 6x
  • 17 µL of digested DNA (K081008 - SpeI+EcoRI) + 3 µL of loading buffer 6x

Migration : 50V, 1h
Image:extractionbefore_biobricks_130710.jpg Image:extractionafter_biobricks_130710.jpg
double digestion of J37033 (LuxR) failed again !

Gel extraction - Kit Qiagen

Ligation (T4 ligase), overnight in cold water
vector : K081008 (LuxI) cut by SpeI + EcoRI
insert : B0015 (ter) cut by EcoRI + XbaI


Aleksandra

Transformation
Transformation into TOP10 - Plating 100µL of each on the plates (Amp) - Incubation overnight :

  • Product of ligation (attC+ter+attC+mRFP)

-> Transformation didn't work, the product of ligation is Kanamycin resistant NOT Ampicillin resistant !!!

DNA Gel electrophoresis (agarose 1,0% w/v) (control of the ligation)
2 µL of EtBr was added in the gel

Protocol (6 wells)

  • 3 µL of ladder 1Kb + T4 ligase (incubation 1h at room temperature)
  • 3 µL of ligation control 1 (vector only) + 3 µL of loading buffer 2x
  • 3 µL of ligation control 2 (vector + ligase) + 3 µL of loading buffer 2x
  • 7 µL of ladder 1Kb
  • 3 µL of ligation product (1 µL) non digested + 3 µL of loading buffer 2x
  • 3 µL of ligation product (1 µL) non digested + 3 µL of loading buffer 2x

Migration : 50V, 30 min
Image:electrophoresis_130710.jpg


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