IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/23: Difference between revisions
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<br><font color=red>Raphaël</font> | <br><font color=red>Raphaël</font> | ||
<br><u>Gel purification</u> with the Quiagen kit. | |||
<br>All bands of the same biobrick are eluted with 30μL of water. | |||
<br><font color=red>Stéphane</font> | |||
<br><u>DNA purification from the 07/23 minipreps</u> using the PCR purification kit. | <br><u>DNA purification from the 07/23 minipreps</u> using the PCR purification kit. | ||
<br>Elution with 30µl water. In the result we have 2x28µl of each biobrick that will serve us as a vector for the ligation: | <br>Elution with 30µl water. In the result we have 2x28µl of each biobrick that will serve us as a vector for the ligation: | ||
*pLux (R0062) | *pLux (R0062) | ||
*attC | *attC | ||
<br><font color=red>Raphaël</font> | |||
<br><u>Minipreps</u> : 4 minipreps of a 5ml culture per biobrick, eluted with 30μL buffer and collected into 1 tube for a total volume of around 120μL: | |||
*attC | |||
*term/attC/mRFP | |||
*attC-term/attC/mRFP (colony from the transformation plate of 07/21) | |||
*pLux-RBS/GFP/term (colony from the transformation plate of 07/21) | |||
<br><u>Nanodrop</u> to determine the amount of DNA in the miniprep samples and after gel purification and PCR purification: | |||
{| | |||
|Sample name || 260/280 ||260/230 ||ng/μL | |||
|- | |||
| attC(miniprep ||1.85 ||2.09||66.6 | |||
|- | |||
| t-attC-mRFP (miniprep) ||1.85||2.17 ||78.3 | |||
|- | |||
| pLux-GFP (miniprep) ||1.86 ||2.38||174.8 | |||
|- | |||
| attC-t-attC-mRFP (miniprep) ||1.83||2.28||75.9 | |||
|- | |||
| attC (PCR purif.) ||1.70 ||1.32||20.0 | |||
|- | |||
| pLux (PCR purif.) ||1.79||1.88||70.3 | |||
|- | |||
| LuxI (gel purif.) ||1.71|| 0.05||5.1 | |||
|- | |||
| LacZ (gel purif.) ||2.26 ||0.02 ||3.1 | |||
|- | |||
| TetA(C) (gel purif.) ||1.92 ||0.08 ||10.8 | |||
|- | |||
| GFP (gel purif.) ||1.80 ||0.08 ||6.2 | |||
|- | |||
| t-attC-mRFP (gel purif.) || 1.40||0.01 ||1.6 | |||
|- | |||
| LacZalpha (I732006) ||1.86 ||2.42 ||221.3 | |||
|- | |||
| LuxR (gel purif.) ||1.66 ||0.06 ||3.4 | |||
|} | |||
<br> Obviously, there is not enough DNA after gel purification to do a ligation... | |||
<br><u>Overnight culture</u> | |||
<br> 2x20mL per biobrick, the goal is to do minipreps on 10mL of pellet tomorrow (increase the initial amount of DNA >> increase the amount of DNA after gel purification !) | |||
*LacZ alpha (I732006) - Amp | |||
*RBS/GFP/term (E0240) - Amp | |||
*LuxR (C0062) - Amp | |||
*RBS/LuxI (K081008) - Amp | |||
*TetA(C) (J31007) - Amp | |||
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Revision as of 14:55, 25 July 2010
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