IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/26: Difference between revisions
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* | <font color=red>Antoine</font> | ||
Electrophoresis of digest products | |||
<br><u>Restriction digest</u> | |||
{| | |||
|Sample name||DNA (3ug)||enzyme||Buffer 2||H2O||BSA | |||
|- | |||
|C0040 || 9.4 ||1 X/P ||2.5||10.85||0.25 | |||
|- | |||
|C0012 || 19.4 ||1 X/P ||2.5||0.85||0.25 | |||
|- | |||
|B0015 || 20.25||1 X/P ||2.5||0||0.25 | |||
|- | |||
|J23110 || 20.25||1 S/P ||2.5||0||0.25 | |||
|- | |||
|P1003 || 6||1 S/P ||2.5||14.25||0.25 | |||
|} | |||
|- | |||
|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> Population counter </span> | |||
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | |||
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<font color=red>Stéphane and Aleksandra</font> | |||
<br><u>Verification of the transformations from 07/23 and 07/22 and if successful preparing them for future ligation</u> | |||
<br><u>Restriction digest of the minipreps from 07/25 and 07/23</u>, incubation at 37°C for 2h | |||
<br>20µl miniprep + 2.5µl Buffer3 10x + 0.25 µl BSA 100x + 1µl XbaI + 1µl PstI (total of 25µl) | |||
*weak RBS (B0031) - TetA(C) (J31007), transformation on 07/23 | |||
*medium RBS (B0032) - TetA(C) (J31007), transformation on 07/23 | |||
*pLux (R0062) - RBS/GFP/term (E0240), transformation on 07/22 | |||
<br>40µl miniprep + 4.7µl Buffer3 10x + 0.5 µl BSA 100x + 2µl XbaI + 2µl PstI (total of 50µl) = double | |||
*weak RBS (B0031) - LuxR (C0062), transformation on 07/23 | |||
*medium RBS (B0032) - LuxR (C0062), transformation on 07/23 | |||
*strong RBS (B0034) - LuxR (C0062), transformation on 07/23 | |||
<br><u>Gel electrophoresis (agarose 1.5% w/v, 50V)</u> | |||
*weak RBS-TetA(C) | |||
*ladder | |||
*medium RBS-TetA(C) | |||
*- | |||
*weak RBS-LuxR | |||
*weak RBS-LuxR | |||
*- | |||
*medium RBS-LuxR | |||
*medium RBS-LuxR | |||
*- | |||
*strong RBS-LuxR | |||
*strong RBS-LuxR | |||
[[Image:gel2307.jpg]][[Image:gel23072.jpg]] | |||
<br>The transformations from 07/23 have worked, we have the inserts of the expected size : 1.2kb for RBS+TetA(C) and 0.8kb for RBS+LuxR. We can purify these inserts to use them in further ligations. | |||
However, the transformation of pLux-RBS/GFP/term from 07/22 didn't work (see the gel made by Théotime further on this page) | |||
<br><u>Gel extraction</u> | |||
<br>Elution from the column with 30µl of warm water (50°C). Samples of the same part are collected into 1 tube. | |||
<br><u>Nanodrop quantification of DNA amounts in the gel extraction samples</u> | |||
{| | |||
|Sample name||260/280||260/230||ng/µl | |||
|- | |||
| || || || | |||
|- | |||
| || || || | |||
|- | |||
|weak RBS-TetA(C) || 1.62 ||0.11 ||11.8 | |||
|- | |||
|medium RBS-TetA(C) ||1.90 ||0.09 ||22.8 | |||
|- | |||
| || || || | |||
|- | |||
|weak RBS-LuxR ||1.76 ||0.38 ||32.3 | |||
|- | |||
|medium RBS-LuxR ||1.89 ||0.08 ||36.8 | |||
|- | |||
| standard RBS-LuxR || 1.80 ||0.16 ||40.7 | |||
|} | |||
<font color=red>Théotime and Raphaël</font> | |||
<br><u>Restriction digest of the biobricks we might need again if the transformations don't work</u> | |||
<br>20µl miniprep + 2.5µl Buffer 10x + 0.25 µl BSA 100x + 1µl enzyme1 + 1µl enzyme2 (total of 25µl) | |||
*RBS/GFP/term (E0240) (XbaI + PstI, B3) | |||
*RBS/LuxI (K081008) (EcoRI-HF + SpeI, B4) | |||
<br>40µl miniprep + 4.7µl Buffer 10x + 0.5 µl BSA 100x + 2µl enzyme1 + 2µl enzyme2 (total of 50µl) = double | |||
*LuxR (C0062) (XbaI + PstI, B3) | |||
*LacZ alpha (XbaI + PstI, B3) | |||
*TetA(C) (XbaI + PstI, B3) | |||
<br><u>Gel electrophoresis (1.5% agarose w/v, 50V)</u> | |||
<br>20µl digestion product + 5µl lb 6x | |||
*ladder 1kb | |||
*RBS/GFP/term | |||
*- | |||
*TetA(C) | |||
*TetA(C) | |||
*- | |||
*LuxR | |||
*LuxR | |||
*- | |||
*- | |||
*- | |||
*pLux-GFP (ligation product purified by Aleksandra and Stéphane) | |||
[[Image:gel2607_2.jpg]][[Image:gel2607_22.jpg]] | |||
<br><u>Gel electrophoresis (3.0% agarose w/v, 50V)</u> | |||
<br>20µl digestion product + 5µl lb 6x | |||
*ladder 0.1kb | |||
*- | |||
*LacZ alpha | |||
*LacZ alpha | |||
*- | |||
*LuxI | |||
[[Image:gel2607_3.jpg]] [[Image:gel2607_32.jpg]] | |||
<br>All of the restictions worked (except for the verification of the ligation pLux-GFP). | |||
<br><u>Gel extraction</u> | |||
<br>Elution from the column with 30µl of warm water (50°C). Samples of the same part are collected into 1 tube. | |||
<br><u>Nanodrop quantification of DNA amounts in the gel extraction samples</u> | |||
{| | |||
|Sample name||260/280||260/230||ng/µl | |||
|- | |||
| || || || | |||
|- | |||
| || || || | |||
|- | |||
| LacZ alpha || 1.94 || 0.02||9.0 | |||
|- | |||
| LuxI||1.82 ||0.05 ||10.2 | |||
|- | |||
| || || || | |||
|- | |||
| || || || | |||
|- | |||
| LuxR || 1.87 ||0.13 ||36.6 | |||
|- | |||
| TetA(C) ||1.88 ||0.37 ||71.8 | |||
|- | |||
| RBS/GFP/term || 1.72 ||0.15 ||15.1 | |||
|} | |||
<br><u>Ligation</u> using around 150ng of the insert and 50µg of the vector, in 10µl of total volume if possible, or in a minimum volume if >10µl. All of the vector DNA (RBSs, Pm, Ps) are diluted 1/5. Incubation at 16°C overnight. | |||
*2µl TetA(C) + 0.9µl stand.RBS + 1µl buffer + 0.5µl ligase + 5.5µl water | |||
*10µl RBS/GFP/term + 3.6µl pLux + 1µl buffer + 0.5µl ligase + 4µl water | |||
*5µl weakRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase | |||
*5µl weakRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 0.8µl water | |||
*4µl medRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase + 1µl water | |||
*4µl medkRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 1.8µl water | |||
*4µl strRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase + 1µl water | |||
*4µl strRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 1.8µl water | |||
*13µl LacZ + 0.7µl weak RBS + 2µl buffer + 0.5µl ligase + 3.8µl water | |||
*13µl LacZ + 0.9µl stand RBS + 2µl buffer + 0.5µl ligase + 3.6µl water | |||
*15µl LuxI + 2.9µl weak RBS + 2µl buffer + 0.5µl ligase | |||
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Revision as of 07:57, 2 August 2010
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Antoine Electrophoresis of digest products
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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> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Stéphane and Aleksandra
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