Team:Paris Liliane Bettencourt/Notebook/2010/07/13/
From 2010.igem.org
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>