IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/22: Difference between revisions
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|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;">Memo cell</span> | ||
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*P1003 LB+Amp+Kan | *P1003 LB+Amp+Kan | ||
*YejF KO (from KEIO collection , KanR disrupt YejF gene) (LB+ Kan)*2 (one for glycerol) | *YejF KO (from KEIO collection , KanR disrupt YejF gene) (LB+ Kan)*2 (one for glycerol) | ||
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|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;">Population counter</span> | |||
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<br><font color=red>Aleksandra, Stéphane, Raphaël</font> | <br><font color=red>Aleksandra, Stéphane, Raphaël</font> | ||
<br><u>Minipreps</u> : 60 minipreps in total, 4 minipreps of a 5ml culture per biobrick, eluted with | <br><u>Minipreps</u> : 60 minipreps in total, 4 minipreps of a 5ml culture per biobrick, eluted with 30μL buffer and collected into 1 tube for a total volume of around 120μL: | ||
* I732006 (LacZ) | * I732006 (LacZ) | ||
* B0031 (weak RBS) | * B0031 (weak RBS) | ||
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* R0062 (pLux) | * R0062 (pLux) | ||
<u>Nanodrop</u> to determine the amount of DNA in the miniprep samples: | <br><font color=red>Aleksandra, Raphaël, Théotime</font> | ||
<br><u>Nanodrop</u> to determine the amount of DNA in the miniprep samples: | |||
{| | {| | ||
|Sample name || 260/280 ||260/230 ||ng/μL | |Sample name || 260/280 ||260/230 ||ng/μL | ||
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|} | |} | ||
<br><font color=red>Aleksandra</font> | |||
<br><u>Restriction digest</u> | <br><u>Restriction digest</u> | ||
<br>This time we use the protocol that says we should use around 3μg of DNA in a minimum amount of water. The volume of the miniprep to use is calculated acording to the nanodrop results. Digestion at 37°C for 2 hours. | <br>This time we use the protocol that says we should use around 3μg of DNA in a minimum amount of water. The volume of the miniprep to use is calculated acording to the nanodrop results. Digestion at 37°C for 2 hours. | ||
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<br><u>Gel electrophoresis (agarose 0.8% w/v), 100V for 30 mins</u> | <br><u>Gel electrophoresis (agarose 0.8% w/v), 100V for 30 mins</u> | ||
<br>This time, gel electrophoresis is used only for the biobricks | <br>This time, gel electrophoresis is used only for the biobricks supposed to be inserts in our constructions, as the cut and the uncut vector biobricks are inseparable on the gel (only several bp are cut out). | ||
We load all the restriction reaction's product into the wells (+6x loading buffer): | We load all the restriction reaction's product into the wells (+6x loading buffer): | ||
*TetA(C), 30μL + 5μL lb | *TetA(C), 30μL + 5μL lb | ||
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*LacZalpha, 30μL + 6μL lb | *LacZalpha, 30μL + 6μL lb | ||
*LacZalpha, 30μL + 6μL lb | *LacZalpha, 30μL + 6μL lb | ||
[[Image:restr2207.jpg]] | |||
[[Image:restr22207.jpg]] | |||
<br> The LacZ alpha is too small and already migrated off the gel, so we couldn't obtain it. The RBS/LuxR biobrick (J370033) still seems to be not working, even though this sample was taken from the 2007 distribution. | |||
<br><u>Gel purification</u> with the Quiagen kit. | |||
<br>All bands of the same biobrick are purified together and eluted with 30μL of water at 50°C. | |||
<br><font color=red>Raphaël</font> | |||
<br><u>PCR purification kit</u> | |||
<br>We simply use the PCR purification kit to purify the vectors for future ligations. Elution with 30μL water. | |||
*stand.RBS (B0034) | |||
*med.RBS (B0032) | |||
*weak RBS (B0031) | |||
*Pstr (J23119) | |||
*Pmed (J23110) | |||
*term (B0015) | |||
<br><u>Nanodrop</u> to determine the amount of DNA after gel purification and PCR purification: | |||
{| | |||
|Sample name || 260/280 ||260/230 ||ng/μL | |||
|- | |||
| || || || | |||
|- | |||
| || || || | |||
|- | |||
| RBS/LuxI (K081009)||1.95 ||0.01 ||1.4 | |||
|- | |||
| LuxR (C0062)||1.73 ||0.10 ||24.5 | |||
|- | |||
| TetA(C) (J31007)|| 1.53|| 0.15||11.0 | |||
|- | |||
| || || || | |||
|- | |||
| || || || | |||
|- | |||
|stand.RBS (B0034) ||1.89 || 2.21||284.8 | |||
|- | |||
|med.RBS (B0032)||1.87 ||2.14 ||236.6 | |||
|- | |||
|weak RBS (B0031) ||1.87 || 2.21||334.5 | |||
|- | |||
| Pstr (J23119)||1.84 || 1.73||91.1 | |||
|- | |||
| Pmed (J23110)||1.85 ||1.84 ||69.6 | |||
|- | |||
| term (B0015)|| 1.82||1.51 ||85.5 | |||
|} | |||
<br><font color=red>Aleksandra</font> | |||
<br><u>Ligation</u> | |||
<br> We used a protocol that advises using 50ng of the vector and 150ng of insert in about 10μL of total volume. Ligation at 16°C overnight. | |||
*8μL LuxR(C0062) + 1μL weak RBS(R0031) diluted 1/6 + 1μL buffer 10x + 0.5μL T4 ligase | |||
*8μL LuxR(C0062) + 1μL med.RBS(R0032) diluted 1/5 + 1μL buffer 10x + 0.5μL T4 ligase | |||
*8μL LuxR(C0062) + 1μL stand.RBS(R0034) diluted 1/5 + 1μL buffer 10x + 0.5μL T4 ligase | |||
*14μL TetA(C) (J31007) + 1μL weak RBS(R0031) diluted 1/6 + 1.7μL buffer 10x + 0.5μL T4 ligase | |||
*14μL TetA(C) (J31007) + 1μL med.RBS(R0033) diluted 1/5 + 1.7μL buffer 10x + 0.5μL T4 ligase | |||
<br><font color=red>Raphaël</font> | |||
<br><u>Overnight culture</u> | |||
<br> 12h for Amp-resistant bacteria, 18h for Kan-resistant bacteria | |||
*attC -Kan | |||
*term/attC/mRFP -Kan | |||
*attC-term/attC/mRFP (colony from the transformation plate of 07/21) -Kan | |||
*pLux-RBS/GFP/term (colony from the transformation plate of 07/21) -Amp | |||
Revision as of 06:31, 29 July 2010
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Antoine
3μL of 1kb Ladder picture of the gel
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Population counter | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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