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
<a href="/wiki/Image:Extractionbefore_biobrick_130710.jpg" class="image" title="Image:extractionbefore_biobrick_130710.jpg"><img alt="Image:extractionbefore_biobrick_130710.jpg" src="/images/f/f1/Extractionbefore_biobrick_130710.jpg" width="355" height="410" border="0" /></a> <a href="/wiki/Image:Extractionafter_biobrick_130710.jpg" class="image" title="Image:extractionafter_biobrick_130710.jpg"><img alt="Image:extractionafter_biobrick_130710.jpg" src="/images/1/15/Extractionafter_biobrick_130710.jpg" width="370" height="406" border="0" /></a>
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
<a href="/wiki/Image:Extractionbefore_biobricks_130710.jpg" class="image" title="Image:extractionbefore_biobricks_130710.jpg"><img alt="Image:extractionbefore_biobricks_130710.jpg" src="/images/5/5d/Extractionbefore_biobricks_130710.jpg" width="362" height="411" border="0" /></a> <a href="/wiki/Image:Extractionafter_biobricks_130710.jpg" class="image" title="Image:extractionafter_biobricks_130710.jpg"><img alt="Image:extractionafter_biobricks_130710.jpg" src="/images/1/13/Extractionafter_biobricks_130710.jpg" width="371" height="410" border="0" /></a>
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
<a href="/wiki/Image:Electrophoresis_130710.jpg" class="image" title="Image:electrophoresis_130710.jpg"><img alt="Image:electrophoresis_130710.jpg" src="/images/a/ab/Electrophoresis_130710.jpg" width="354" height="407" border="0" /></a>