Team:UNAM Genomics Mexico/Notebook/OR

From 2012.igem.org

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__NOTOC__
<br />
<br />
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<h1>Under Construction</h1>  
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<center><h1>'''Or Gate'''</h1></center>
<br />
<br />
<br />
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<html>
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<table border="0"  height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg" cellpadding="20">
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<tr>
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<td id="leftcolumn2"><center>2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JUNE | '''JUNE''']] </center> <br />
 +
<br />
 +
2.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F07.2F12 |06/07/12]] <br />
 +
2.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F12.2F12 |06/12/12]] <br />
 +
2.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F13.2F12 |06/13/12]] <br />
 +
2.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F14.2F12 |06/14/12]] <br />
 +
2.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F15.2F12 |06/15/12]] <br />
 +
2.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F18.2F12 |06/18/12]] <br />
 +
2.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F19.2F12 |06/19/12]] <br />
 +
2.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F22.2F12 |06/22/12]] <br />
 +
2.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F25.2F12 |06/25/12]] <br />
 +
2.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F26.2F12 |06/26/12]] <br />
 +
2.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F27.2F12 |06/27/12]] <br />
 +
2.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#06.2F29.2F12 |06/29/12]] <br />
 +
</td>
 +
 
 +
<td id="contentcolumn2"><center>3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#JULY | '''JULY''']] </center> <br />
 +
<br />
 +
3.1[[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F03.2F12 | 07/03/12]] <br />
 +
3.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F04.2F12 | 07/04/12]] <br />
 +
3.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F06.2F12 | 07/06/12]] <br />
 +
3.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F07.2F12 | 07/07/12]] <br />
 +
3.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F08.2F12 | 07/08/12]] <br />
 +
3.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F09.2F12 | 07/09/12]] <br />
 +
3.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F10.2F12 | 07/10/12]] <br />
 +
3.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F11.2F12 | 07/11/12]] <br />
 +
3.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F12.2F12 | 07/12/12]] <br />
 +
3.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F13.2F12 | 07/13/12]] <br />
 +
3.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F14.2F12 | 07/14/12]] <br />
 +
3.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F16.2F12 | 07/16/12]] <br />
 +
3.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F17.2F12 | 07/17/12]] <br />
 +
3.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F18.2F12 | 07/18/12]] <br />
 +
3.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F19.2F12 | 07/19/12]] <br />
 +
3.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F20.2F12 | 07/20/12]] <br />
 +
3.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F23.2F12 | 07/23/12]] <br />
 +
3.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F24.2F12 | 07/24/12]] <br />
 +
3.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F25.2F12 | 07/25/12]] <br />
 +
3.20 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F26.2F12 | 07/26/12]] <br />
 +
3.21 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F27.2F12 | 07/27/12]] <br />
 +
3.22 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F30.2F12 | 07/30/12]] <br />
 +
3.23 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#07.2F31.2F12 | 07/31/12]] <br />
 +
</td>
 +
 
 +
<td id="rightcolumn2"><center>4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#AUGUST | '''AUGUST''']] </center> <br />
 +
<br />
 +
4.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F01.2F12 | 08/01/12]] <br />
 +
4.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F02.2F12 | 08/02/12]] <br />
 +
4.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F03.2F12 | 08/03/12]] <br />
 +
4.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F06.2F12 | 08/06/12]] <br />
 +
4.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F08.2F12 | 08/08/12]] <br />
 +
4.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F09.2F12 | 08/09/12]] <br />
 +
4.7 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F10.2F12 | 08/10/12]] <br />
 +
4.8 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F13.2F12 | 08/13/12]] <br />
 +
4.9 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F16.2F12 | 08/16/12]] <br />
 +
4.10 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F20.2F12 | 08/20/12]] <br />
 +
4.11 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F21.2F12 | 08/21/12]] <br />
 +
4.12 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F22.2F12 | 08/22/12]] <br />
 +
4.13 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F23.2F12 | 08/23/12]] <br />
 +
4.14 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F24.2F12 | 08/24/12]] <br />
 +
4.15 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F25.2F12 | 08/25/12]] <br />
 +
4.16 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F28.2F12 | 08/28/12]] <br />
 +
4.17 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F29.2F12 | 08/29/12]] <br />
 +
4.18 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F30.2F12 | 08/30/12]] <br />
 +
4.19 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#08.2F31.2F12 | 08/31/12]] <br />
 +
</td>
 +
</tr>
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<table border="0"  height="150" cellspacing="15" bgcolor="transparent" id="tablecontentbg">
 
<tr>
<tr>
-
<td id="leftcolumn2">Nanotubes!!</td>
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<td id="leftcolumn2"><center>5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#SEPTEMBER | '''SEPTEMBER''']] </center> <br />
-
<td  id="contentcolumn2">The logic</td>
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<br />
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<td id="rightcolumn2">Random info</td>
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5.1 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F01.2F12 | 09/01/12]] <br />
 +
5.2 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F02.2F12 | 09/03/12]] <br />
 +
5.3 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F04.2F12 | 09/04/12]] <br />
 +
5.4 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F05.2F12 | 09/05/12]] <br />
 +
5.5 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F06.2F12 | 09/06/12]] <br />
 +
5.6 [[Team:UNAM_Genomics_Mexico/Notebook/ANDSugar#09.2F14.2F12 | 09/14/12]] <br />
 +
 
 +
</td>
 +
 
 +
<td  id="contentcolumn2"><center>[[File:UnmagenomicsBacteria sugar.png | 190px]]</center>
 +
</td>
 +
 
 +
<td id="rightcolumn2"><p> '''On hover the images to see descriptions'''</p><td/>
</tr>
</tr>
</table>
</table>
 +
 +
 +
=JUNE=
 +
<br />
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_1.jpg' height="300"/>
 +
<div class='captionnaranja'>
 +
<p class='captionInside'>1. 1 kb ladder <br />
 +
2.E1010
 +
</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<h2>06/07/12</h2><br />
 +
PY BROTH 10g salt/L<br />
 +
PSB2K3 kanamicin<br />
 +
We transformed RFP E1010. <br />
 +
plate 1 18F<br />
 +
plate 2 17E<br />
 +
Stock 50 mg/mL<br />
 +
E.coli 1/1000<br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
We made liquid cultures with colonies [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
We extracted plasmids [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PLASMID_EXTRACTION_2 | PLASMID  EXTRACTION PROTOCOL]]. <br />
 +
We ran a gel to check the extraction [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br />
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
<h2>06/12/12</h2><br />
 +
We made glycerols of the bacteria <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br />
 +
We transformed plasmid pHp45 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/3/38/UnamgenomicsandsugarBitacora_2.jpg' height="300"/>
 +
<div class='captiongray'>
 +
<p class='captionInside'>1. 1 kb ladder <br />
 +
2.E1010
 +
</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
</html>
</html>
 +
<h2>06/13/12</h2><br />
 +
We ran gel and extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br />
 +
We made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] and lysed [LYSIS PROTOCOL]. <br />
 +
<br />
 +
<br />
 +
<br />
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<br />
 +
<br />
 +
<br />
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<br />
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<br />
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<br />
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<br />
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 +
<br />
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<br />
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 +
<br />
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/2/2d/UnamgenomicsandsugarBitacora_3.jpg' height="300"/>
 +
<div class='captionrojo'>
 +
<p class='captionInside'>1. 1 kb ladder <br />
 +
We extracted from gel
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<h2>06/14/12</h2><br />
 +
We extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]].<br />
 +
We made glycerols of pHp45 Ω [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br />
 +
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450
 +
PSB4A5 Am (ampicillin) 1I BBa_J04450
 +
AraC BBa_C0080 <br />
 +
2012 14L plate 1 pSB2K3 Km+<br />
 +
2011 14L plate 1 pSB2K3 Km+<br />
 +
2012 14L plate 1 pSB2K3 Km+<br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
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<br />
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<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/5/56/UnamgenomicsandsugarBitacora_5.jpg' height="300"/>
 +
<div class='captionverde'>
 +
<p class='captionInside'>1. 1 kb ladder <br />
 +
pHp45 Ω with E/P<br />
 +
We extracted from gel<br />
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<h2>06/15/12</h2><br />
 +
> Digested pHp45 Ω with E/P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
> We extracted plasmid with kit. <br />
 +
> Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (5) <br />
 +
>Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
<br />
 +
ARAC<br />
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>From the overnight plate we prepared liquid culture [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
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>We left them incubating overnight. <br />
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>We left a plate (LB Km DH5α C0080 and a control). <br />
 +
<br />
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<br />
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<br />
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<br />
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<br />
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<h2>06/18/12</h2><br />
 +
>Plasmid extraction AraC with kit. <br />
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>C0080-psB2K3 915 bp<br />
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>Streaked 4 LB Km 30 plates DH5α psB2K3. <br />
 +
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red. <br />
 +
>Ran gel with Spr/Strr (eppendorf -20ºC) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br />
 +
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04. <br />
 +
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]. <br />
 +
>Digested C0080 X,S. <br />
 +
>Digested B0014 E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
<br />
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/5/52/UnamgenomicsandsugarBitacora_6.jpg' height="300"/>
 +
<div class='captionrosa'>
 +
<p class='captionInside'>1. 1 kb ladder <br />
 +
Digested B0014 with E, P and with E,X
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<br />
 +
<br />
 +
 +
<h2>06/19/12</h2><br />
 +
>Digested B0014 with E, P and with E,X. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
>Ran gels with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation. <br />
 +
C0080 is in the first lane. <br />
 +
>Extracted from gel C0080 X,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
>Made glycerols from 4 plates LB Km DH5α pSB2K3 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br />
 +
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br />
 +
AmyE 5’ 18K plate 3 2010, 2011, 2012<br />
 +
AmyE 3’ 18M plate 3 2010, 2011, 2012<br />
 +
>Digested B0014 with E,X and E,P again [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
<br />
 +
<br />
 +
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<br />
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<br />
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 +
<br />
 +
<br />
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 +
<h2>06/22/12</h2><br />
 +
 +
>Ran gels with digestions B0014 with E,X and E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]. <br />
 +
 +
>Dephosphated B0014 E,X and B0014 E,P [DESPHOPHORYLATION PROTOCOL]. <br />
 +
<br />
 +
 +
<br />
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 +
<br />
 +
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<br />
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<h2>06/25/12</h2><br />
 +
>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS]. <br />
 +
 +
<h2>06/26/12</h2><br />
 +
>Ran gel with psB2K3 and psB4A5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
 +
<h2>06/27/12</h2><br />
 +
>Transformed with plasmid B0079 1576 bp psB1A2 12A [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011<br />
 +
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011<br />
 +
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011<br />
 +
AmyE 5’ grew 2 colonies. <br />
 +
 +
<h2>06/29/12</h2><br />
 +
>We did a DH5α K143001 Km30 Amp 100 glycerol [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]]  07. <br />
 +
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control<br />
 +
These were both plated in 2 plates each. <br />
 +
They were left overnight since they had not grown by 7:00 pm so  the next day glycerols were made. <br />
 +
>Liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
 +
2 tubes DH5α K143001 Km30 Amp 100 <br />
 +
2 tubes DH5α K143002 Km30 Amp 100 <br />
 +
2 tubes DH5α B0079 Amp 100 <br />
 +
1 tube LB Km 30 Amp 100 control<br />
 +
1 tube LB Amp 100 control<br />
 +
>From the 6 tubes we extracted plasmid from kit. <br />
 +
<br />
 +
<br />
 +
=JULY=
 +
 +
07/02/12<br />
 +
>Digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
B0079 digestion with S,P<br />
 +
K143001 with S,P<br />
 +
K143002 with S,P <br />
 +
>PCR’s<br />
 +
•AraC<br />
 +
•Cassete  ΩSpr/Strr  <br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#PCR_Protocol_.2850.C2.B5l.29 | PCR PROTOCOL]] <br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/b/be/UnamgenomicsandsugarBitacora_8.jpg' height="300"/>
 +
<div class='captionmoradoclaro'>
 +
<p class='captionInside'>After Cassete  ΩSpr/Strr  PCR we ran a gel
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/c/cd/UnamgenomicsandsugarBitacora_9.jpg' height="300"/>
 +
<div class='captionmorado'>
 +
<p class='captionInside'>B0079 digestion with S,P<br />
 +
K143001 with S,P<br />
 +
K143002 with S,P <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>07/03/12</h2><br />
 +
>After Cassete  ΩSpr/Strr  PCR we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (8) <br />
 +
>Ran gel with digestions from yesterday. (9) <br />
 +
>Did band extractions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
>Stored at -20ºC. <br />
 +
>Did PCR to Cassete  ΩSpr/Strr  to add RBS and prefix/suffix [PCR PROTOCOL]. <br />
 +
>Left digesting with E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/0/03/UnamgenomicsandsugarBitacora_9.1.jpg' height="300"/>
 +
<div class='captionazul'>
 +
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>07/04/12</h2><br />
 +
>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1) [DESPHOPHORYLATION PROTOCOL]. <br />
 +
>Ligated Cassete  ΩSpr/Strr  +K143002 dephosphated(digested with E,X and E,S) LIGATION PROTOCOL]. <br />
 +
>Transformed ligation and left overnight plated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
>Digested with PstI [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<h2>07/06/12</h2><br />
 +
4 LB Km30 Spec60 DH5α  ΩSpr/Strr  +K143002  still have not grown. <br />
 +
Ran a gel with yesterday’s digestions to chek if they were done properly (10) <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
Ran another gel with the rest of the samples. <br />
 +
Extracted GusA fragment <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
Repeated ligation  ΩSpr/Strr  +K143002 E,X dephosphated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
Repeated  ΩSpr/Strr  PCR. <br />
 +
<br />
 +
<br />
 +
<h2>07/07/12</h2><br />
 +
Transformed DH5α  ΩSpr/Strr  +K143002  ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
Plated LB Km30 Spec 60 DH5α  ΩSpr/Strr  +K143002 . <br />
 +
<br />
 +
<br />
 +
 +
<h2>07/08/12</h2><br />
 +
Transformed DH5α  ΩSpr/Strr  +K143002  ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
Plated LB Km30 Spec 60 DH5α  ΩSpr/Strr  +K143002. <br />
 +
<br />
 +
<br />
 +
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/1/13/UnamgenomicsandsugarBitacora_11.jpg' height="300"/>
 +
<div class='captiongray'>
 +
<p class='captionInside'>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/b/b2/UnamgenomicsandsugarBitacora_11.1.jpg' height="300"/>
 +
<div class='captionnaranja'>
 +
<p class='captionInside'>1 PCR omega P<br />
 +
2 PCR omega I<br />
 +
3 PCR AraC P<br />
 +
4 PCR AraC I<br />
 +
5 ladder 1 Kb<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<h2>07/09/12</h2><br />
 +
From yesterday’s transformations only one colony grew. <br />
 +
From the previous transformation only 2 colonies grew. <br />
 +
These 3 were streaked in 3 plates: <br />
 +
DH5α  ΩSpr/Strr  +K143002  LB Km 30 Sp 100 1. <br />
 +
DH5α  ΩSpr/Strr  +K143002  LB Km 30 Sp 100 2. <br />
 +
DH5α  ΩSpr/Strr  +K143002  LB Km 30 Sp 100 3. <br />
 +
LB Km 30 Sp 100 control. <br />
 +
Did liquid cultures in 3 tubes: <br />
 +
ΩSpr/Strr  +K143002  LB Km 30 Sp 100 1. <br />
 +
ΩSpr/Strr  +K143002  LB Km 30 Sp 100 2. <br />
 +
ΩSpr/Strr  +K143002  LB Km 30 Sp 100 3. <br />
 +
LB Km30 Sp 100 control. <br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
 +
Ran a gel with: (11) <br />
 +
GusA P<br />
 +
PBBR1 GusA<br />
 +
Ω E PCR P<br />
 +
Ω PCR P<br />
 +
Ω PCR I<br />
 +
Ω E<br />
 +
Ω PCR I<br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] <br />
 +
Did GusA primers dissolution for PCR. <br />
 +
GusA PCR <br />
 +
[PCR PROTOCOL] <br />
 +
Gel Extraction by kit of lanes 3 and 5 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
 +
'''PCR omega P, PCR omega I, PCR AraC P, PCR AraC I'''<br />
 +
Add 10 μl  of each primer (LW and UP). <br />
 +
Add 3 μl  of plasmid (P). <br />
 +
Add 30.4 μl  buffer. <br />
 +
Add 5 μl  Mg. <br />
 +
Add 8 μl  DNTp’s. <br />
 +
Add 42.6μl  H2O miliQ. <br />
 +
Add 1 μl  RTTG polymerase. <br />
 +
Centrifuge (spin) 8 secs. <br />
 +
Add vegetable oil till the eppendorf is full. <br />
 +
Place eppendorf 1 mL in thermocycler. <br />
 +
Run PCR with program “BERNA”. <br /><br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/4/4b/UnamgenomicsandsugarBitacora_11.2.jpg' height="300"/>
 +
<div class='captionverde'>
 +
<p class='captionInside'>GusA digestions with X,P;  Ω+AmyE 3’ with E,X; AraC PCR with E,S <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>07/10/12</h2><br />
 +
•GusA digestions with X,P;  Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times)[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTIONS]]. (11.2) <br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<h2>07/11/12</h2><br />
 +
•GusA PCR digestions with E,S;  Ω+AmyE 3’ with E,X [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
•T.V. AraC PCR+ Ω+AmyE 3’  E,X dephosphated ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/0/0a/UnamgenomicsandsugarBitacora_12.jpg' height="300"/>
 +
<div class='captionrojo'>
 +
<p class='captionInside'>1. ladder. <br />
 +
2. GusA PCR E,S. <br />
 +
3. Ω+AmyE 3’ with E,X. <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>07/12/12</h2><br />
 +
•Ran a gel with yesterday’s digestions: (12) <br />
 +
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
 +
•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
 +
•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
•Made glycerols from 2 LB Km 30 Sp100 DH5α  Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br />
 +
•Ligated GusA PCR with B0014 E,X desphophorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
•Digested Pfrc54 (A3) with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
 +
•Desphophorylated  Ω+AmyE 3’ E,X [DESPHOPHORYLATION PROTOCOL]. <br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/0/0f/UnamgenomicsandsugarBitacora_13.jpg' height="300"/>
 +
<div class='captionrojo'>
 +
<p class='captionInside'>•Ran gel with pfrc54 S,P<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>07/13/12</h2><br /><br />
 +
•Ran gel with pfrc54 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (13) <br />
 +
•Transformation of GusA PCR + B0014 ligation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
•Transformed with GFP E0040 psBIA2. <br />
 +
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
•B. Subtitils competent cells [''B.Subtilis'' group protocol]. <br />
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
<br />
 +
 +
<br />
 +
<h2>07/14/12</h2><br /><br />
 +
•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes: <br />
 +
•Extracted pellets<br />
 +
•Extracted plasmids [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
•Ran gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br /><br />
 +
•From transformed DH5α km 30: <br />
 +
GusA+B0014 DH5α Km50 24 pellets <br />
 +
E0040 DH5α LB Amp100 <br />
 +
From these two we: <br />
 +
•Did liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
•Extracted plasmid [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid  [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
•Ran gel with this transformation [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
<br />
 +
<br />
 +
<br />
 +
<h2>07/16/12</h2><br /><br />
 +
•Digested E,P AraC+ Ω+AmyE 3’ <br />
 +
•Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
<br />
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/4/48/UnamgenomicsandsugarBitacora_14.jpg' height="300"/>
 +
<div class='captionrosa'>
 +
<p class='captionInside'>Digested E,P AraC+ Ω+AmyE 3’ <br />
 +
Ω+AmyE 3’ E,P <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<h2>07/17/12</h2><br /><br />
 +
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (14) <br />
 +
•The gel we ran didn’t work, probably because the agarose was not prepared correctly. <br />
 +
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
<br />
 +
<br />
 +
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/d/db/Bitacora_15.jpg' height="300"/>
 +
<div class='captionaqua'>
 +
<p class='captionInside'>•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<br />
 +
 +
<h2>07/18/12</h2><br /><br />
 +
•From yesterday’s digestions we ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (15) <br /><br />
 +
•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br /><br />
 +
•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
 +
•We digested 1 + Ω+AmyE 3’ E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
 +
 +
<h2>07/19/12</h2><br /><br />
 +
•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
 +
•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br /><br />
 +
•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21 [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br /><br />
 +
•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
 +
<br />
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/f/f3/UnamgenomicsandsugarBitacora_16.jpg' height="300"/>
 +
<div class='captionmoradoclaro'>
 +
<p class='captionInside'>Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>07/20/12</h2><br /><br />
 +
•Ran gel with yesterday’s digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . (16) <br />
 +
•Transformed GusA+B0014 in two tubes [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
<h2>07/23/12</h2><br /><br />
 +
•From 24 GusA+B0014 tubes (-) we didn’t do anything. <br />
 +
•From 4 AraC+ Ω+AmyE 3’  we extracted plasmid [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
•Digested from the 4 AraC+ Ω+AmyE 3’  E,X; X; P and GusA PCR with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
 +
 +
<h2>07/24/12</h2><br /><br />
 +
•Digested B0014 E with X
 +
B0014 X with E<br />
 +
•Digested AraC+ Ω+AmyE 3’  1,2,3,4 with E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
 +
<h2>07/25/12</h2><br /><br />
 +
•Ran gel of digested AraC+ Ω+AmyE 3’  1,2,3,4 with E,P  [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
•Joined B0014 E with X B0014 X with E digestions. <br />
 +
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. Transformed [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
•From LasR DH5α make liquid cultures and plate [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
•Digested  Ω+AmyE 3’  with X,P; E,S; C0080 X,P; E,S; S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
 +
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/8/84/UnamgenomicsandsugarBitacora_17.jpg' height="300"/>
 +
<div class='captionazul'>
 +
<p class='captionInside'>1.Ω+AmyE 3’ X,P<br />
 +
2. Ω+AmyE 3’ E,S<br />
 +
3.C0080 X,P<br />
 +
4.C0080 E,S<br />
 +
5.C0080 S,P<br />
 +
6.ladder<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>07/26/12</h2><br />
 +
•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
 +
•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
 +
•Due to problems with the way we did the transformations of ligations we repeated them: <br />
 +
GusAPCR X,P+ B0079 S,P dephosphorylated<br />
 +
 +
GusAPCR X,P+ B0079 S,P dephosphorylated (-)<br />
 +
 +
GusAPCR X,P+ A3 S,P dephosphorylated<br />
 +
 +
GusAPCR X,P+ A3 S,P dephosphorylated (-)<br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
 +
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
•AraC+ Ω+AmyE 3’  S,P 1,2,3,4<br />
 +
•K143002 X,P<br />
 +
•AraC+ Ω S,P<br />
 +
•C0179 X,S<br />
 +
 +
 +
<h2>07/27/12</h2><br /><br />
 +
Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
<h2>07/30/12</h2><br /><br />
 +
•Transformed with ligations: <br />
 +
•AraC+ Ω  dephosphprylated +K143002 X,P<br />
 +
•GusAPCR X,P+ A3 S,P dephosphorylated<br />
 +
•GusAPCR X,P+ B0079 S,P dephosphorylated<br />
 +
•Transformed the following sythesis: <br />
 +
•91996 Pveg 140 bp<br />
 +
•91997 ArsR-CzrA_promoter 1 194 bp<br />
 +
•91998 ArsR-CzrA_promoter 2 221 bp<br />
 +
•91999 ArsR-CzrA_promoter 3 213 bp<br />
 +
•92000 pBad-pXyl 387 bp<br />
 +
•92001 XylR 1117pb<br />
 +
•92002 CI_pro_(NAND_INHIBITOR) 774 <br />
 +
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked.
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]]. <br />
 +
 +
•Make liquid cultures of the following transformations for tomorrow [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]: <br />
 +
•AraC+ Ω+K143002 <br />
 +
•GusA+A3<br />
 +
•GusA+B0079<br />
 +
•Synthesis<br />
 +
 +
<h2>07/31/12</h2><br /><br />
 +
Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
<br />
 +
<br />
 +
 +
=AUGUST=
 +
 +
<h2>08/01/12</h2><br /><br />
 +
Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br /><br />
 +
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/b/b1/UnamgenomicsandsugarBitacora_17.1.jpg' height="300"/>
 +
<div class='captiongray'>
 +
<p class='captionInside'>1 PCR GusA I<br />
 +
2 PCR GusA I<br />
 +
3 PCR GusA P<br />
 +
4 PCR GusA P<br />
 +
5 ladder 1 Kb<br /></p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>08/02/12</h2><br /><br />
 +
•Extracted plasmids from liquid cultures[PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]: <br />
 +
AraC+ Ω+K143002 <br />
 +
•GusA+A3<br />
 +
•GusA+BBR1<br />
 +
 +
PCR GusA, GusA I, PCR GusA P, PCR GusA I<br />
 +
 +
•Add 10 μl  of each primer (LW and UP). <br />
 +
•Add 3 μl  of plasmid (P). <br />
 +
•Add 30.4 μl  buffer. <br />
 +
•Add 5 μl  Mg. <br />
 +
•Add 8 μl  DNTp’s. <br />
 +
•Add 42.6μl  H2O miliQ. <br />
 +
•Add 1 μl  RTTG polymerase. <br />
 +
•Centrifuge (spin) 8 secs. <br />
 +
•Add mineral oil till the eppendorf is full. <br />
 +
•Place eppendorf 1 mL in thermocycler. <br />
 +
•Run PCR with program “BERNA”. <br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/8/82/UnamgenomicsandsugarBitacora_18.jpg' height="300"/>
 +
<div class='captionnaranja'>
 +
<p class='captionInside'>1.GusA PCR 1<br />
 +
2.GusA PCR 2<br />
 +
3.A3+GusA 1<br />
 +
4.A3+GusA 2<br />
 +
5.A3+GusA 3<br />
 +
6.P GusA<br />
 +
7.P GusA 2<br />
 +
8.Ladder<br />
 +
9.01<br />
 +
10.06<br />
 +
11.96<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<br />
 +
<br />
 +
 +
<h2>08/03/12</h2><br /><br />
 +
• Ran a gel with: (18) <br />
 +
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
•Ran another gel to extract with: <br />
 +
1.GusA PCR 1<br />
 +
2.GusA PCR 2<br />
 +
3.00<br />
 +
4.01<br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
•Did the following digestions [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]: <br />
 +
 +
• Ω+K143002  E,X; A3+GusA E,S; GusAPCR X,P <br />
 +
 +
•Extracted GusAPCR X,P  digestion [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
 +
•Left the following ligation: AraC+ Ω+K143002  [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br /><br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/1/17/UnamgenomicsandsugarBitacora_19.jpg' height="300"/>
 +
<div class='captionrojo'>
 +
<p class='captionInside'>1.A3+GusA 2<br />
 +
2.A3+GusA 3<br />
 +
3. Ω+AmyE 3’ 2<br />
 +
4. Ω+AmyE 3’ 3<br />
 +
5.XylR X,P<br />
 +
6.pBad-pXyl X,P<br />
 +
7.GusA PCR X,P<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
 +
<h2>08/06/12</h2><br /><br />
 +
•Made liquid cultures of  Ω+K143002  (2 cultures+1 control) and AraC+ Ω+K143002  (2 cultures+1 control) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
•Ran a Gel with: (19) <br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/8/8b/UnamgenomicsandsugarBitacora_19.1.jpg' height="300"/>
 +
<div class='captionverde'>
 +
<p class='captionInside'>Digested 14 tubes of AraC+ Ω+AmyE 3’  E,P <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>08/08/12</h2><br />
 +
BBa_B0040 6I psB1A2 Amp+ plate 1<br />
 +
Digested 14 tubes of AraC+ Ω+AmyE 3’  E,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (19.1) <br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<h2>08/09/12</h2><br />
 +
•From yesterday’s PCR’s : <br />
 +
• E0040 plasmid 1<br />
 +
E0040 plasmid 2<br />
 +
E0040 digested 1<br />
 +
E0040 digested 2<br />
 +
We purified with PCR kit<br />
 +
 +
•Ran  a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
•Made 14 PCR’s from AraC+ Ω+AmyE 3’. <br />
 +
•From B0079+GusA ligation and B0040 transformation:
 +
•Grew colonies. <br />
 +
•Made liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
•Streaked these in a new plate. <br />
 +
•Diluted plasmid E0080 2 1/50. <br />
 +
<br />
 +
 +
<br />
 +
 +
<h2>08/10/12</h2><br />
 +
•Yesterday’s gel did not come out as expected so we repeated the PCR. <br />
 +
<br />
 +
 +
<br />
 +
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/e/ef/UnamgenomicsandsugarBitacora_20.jpg' height="300"/>
 +
<div class='captionrosa'>
 +
<p class='captionInside'>1.AraC+ Ω+AmyE 3’  <br />
 +
2-14.AraC+ Ω+AmyE 3’  <br />
 +
15.AraC+ Ω 1<br />
 +
16.AraC+ Ω 1<br />
 +
17.AraC+ Ω 1<br />
 +
18.PCR 1 GFP<br />
 +
19.PCR GFP<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<br />
 +
<h2>08/13/12</h2><br />
 +
•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’  and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (20) <br />
 +
 +
•Extracted pasmids form liquid cultures: B0049, B0079+GusA [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
 +
•AraC+ Ω+AmyE 3’  undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω<br />
 +
 +
• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.
 +
 +
• We ran a gel to extract. <br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/f/f1/UnamgenomicsandsugarBitacora_22.jpg' height="300"/>
 +
<div class='captionaqua'>
 +
<p class='captionInside'>1.6 AraC+ Ω+AmyE 3’  E  with P. <br /><br />
 +
2.8 AraC+ Ω+AmyE 3’  E  with P. <br />
 +
3.11 AraC+ Ω+AmyE 3’  E  with P. <br />
 +
4.12 AraC+ Ω+AmyE 3’  E  with P. <br />
 +
5.6 AraC+ Ω+AmyE 3’  with E,X. <br />
 +
6.8 AraC+ Ω+AmyE 3’  with E,X. <br />
 +
7.11 AraC+ Ω+AmyE 3’  with E,X. <br />
 +
8.12 AraC+ Ω+AmyE 3’  with E,X. <br />
 +
9.00 pBad/pXyl with S,P. <br />
 +
10.Ladder. <br />
 +
11.E0040 PCR E,S to extract the correct band. <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
 +
<h2>08/16/12</h2><br />
 +
•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (22)PROTOCOL]: <br />
 +
 +
•Digested: <br />
 +
ArSR-CzrA 97 with S,P<br />
 +
ArSR-CzrA 98 with S,P<br />
 +
ArSR-CzrA 99 with S,P<br />
 +
E0040 PCR with E,S<br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
 +
<h2>08/20/12</h2><br />
 +
•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
 +
<h2>08/21/12</h2><br />
 +
•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated [DEPHOSPHORYLATION PROTOCOL]. <br />
 +
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
•Digested  Ω+AmyE 3’  with E,X and + Ω+AmyE 3’  II with E,X (obtained these two from glycerols stored at -80ºC [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
 +
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/2/29/UnamgenomicsandsugarBitacora_22.1.jpg' height="300"/>
 +
<div class='captionmoradoclaro'>
 +
<p class='captionInside'>Digested K143001+PBad, pXyl with E,S <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/1/1c/UnamgenomicsandsugarBitacora_23.jpg' height="300"/>
 +
<div class='captionmorado'>
 +
<p class='captionInside'>1.K143001+PBad, pXyl  E,S 10<br />
 +
2.K143001+PBad, pXyl  E,S 11<br />
 +
3.K143001+PBad, pXyl  E,S 12<br />
 +
4.A3 PCR<br />
 +
5.A3 PCR<br />
 +
6.E0040 PCR E,S<br />
 +
7.B0014 E,X dephosphorylated<br />
 +
8. Ω+AmyE 3’  E,X <br />
 +
9. Ω+AmyE 3’  E,X II<br />
 +
10.Ladder
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>08/22/12</h2><br />
 +
•We did 2 PCR’s for A3  [PCR PROTOCOL]. <br />
 +
 +
•Digested K143001+PBad, pXyl with E,S <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. (22.1) <br />
 +
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
•Did band extraction of: <br />
 +
1. K143001+PBad, pXyl  E,S 10<br />
 +
2.K143001+PBad, pXyl  E,S 11<br />
 +
3.K143001+PBad, pXyl  E,S 12<br />
 +
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br />
 +
 +
•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’  6,8,11,12 Km+ and Spectinomycin+ [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#GLYCEROL_PROTOCOL | GLYCEROL PROTOCOL]] . <br />
 +
 +
•Transformed ligations: <br />
 +
B0079+GusA Amp+<br />
 +
Pveg+XylR Chloramphenicol+
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Transformation | TRANSFORMATION PROTOCOL]].. <br />
 +
<br />
 +
 +
<br />
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/7/78/UnamgenomicsandsugarBitacora_23.01.jpg' height="300"/>
 +
<div class='captionazul'>
 +
<p class='captionInside'>1. Ω+AmyE 3’  E,X 6.2<br />
 +
2.Ω+AmyE 3’  II E,X<br />
 +
3.97 ArsR-CzrA<br />
 +
4.98 ArsR-CzrA<br />
 +
5.99 ArsR-CzrA
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>08/23/12</h2><br />
 +
•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this. <br />
 +
•Ran gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | Digestion Protocol]]. <br />
 +
 +
 +
<h2>08/24/12</h2><br />
 +
From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’  6 and 8  we streaked 2 plates for glycerol of each ligation. <br />Repeated ligation B0079+GusA. <br />
 +
 +
Ligated E0040 PCR E,S+B0014 E,X dephospohylated [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
Made 2 new plates from Pveg+XylR ligation and liquid cultures [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
 +
 +
<h2>08/25/12</h2><br />
 +
Extracted pellet from 16 tubes of pVeg+XylR that were left overnight [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
 +
Ligated B0079+GusA and B0040+B0014 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
 +
<h2>08/28/12</h2><br />
 +
 +
Made liquid cultures from transformations tht were left overnight
 +
-R0079+GusA<br />
 +
-R0079+GusA (-)<br />
 +
-E0040+B0014<br />
 +
-E0040+B0014 (-)<br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/2/25/UnamgenomicsandsugarBitacora_23.1.jpg' height="300"/>
 +
<div class='captiongray'>
 +
<p class='captionInside'>1. A3 PCR 1. <br />
 +
2. A3 PCR 2. <br />
 +
3. Ω+AmyE 3’  S,P *.<br />
 +
4. Ω+AmyE 3’  S,P . <br />
 +
5. Pveg+XylR E,S 2. <br />
 +
6. Pveg+XylR E,S 3. <br />
 +
7. Pveg+XylR E,S 4. <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>08/29/12</h2><br />
 +
Digested Pveg+XylR 2,3,4 with E,S. <br />
 +
 +
Digested  Ω+AmyE 3’  with S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | DIGESTION]]. <br />
 +
 +
Ran a PCR with A3 PCR (2 .6 ml tubes) [PCR PROTOCOL]. <br />
 +
 +
Ran a gel with <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (23.01) <br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/2/2e/UnamgenomicsandsugarBitacoraBitacora_24.jpg' height="300"/>
 +
<div class='captiongray'>
 +
<p class='captionInside'>1-23. E0040 + B0014 colonies<br />
 +
24-29. B0079+GusA 1<br />
 +
30. B0079<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>08/30/12</h2><br />
 +
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (24) <br />
 +
 +
 +
•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/3/3d/UnamgenomicsandsugarBitacora_25.jpg' height="300"/>
 +
<div class='captionnaranja'>
 +
<p class='captionInside'>1-3B0079+GusA E,S 3. <br />
 +
4-9. PVeg+XylR  E,S 1. <br />
 +
10-17. E0040+B0014 X,P 3. <br />
 +
18. 1 kb ladder. <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/d/d2/UnamgenomicsandsugarBitacora_26.jpg' height="300"/>
 +
<div class='captionrojo'>
 +
<p class='captionInside'>1.A3 PCR. <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/7/7b/UnamgenomicsandsugarBitacora_27.jpg' height="300"/>
 +
<div class='captionverde'>
 +
<p class='captionInside'>E0040+B0014 X,P 4 to extract<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<h2>08/31/12</h2><br />
 +
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (25) <br />
 +
 +
 +
•Dephosphorylated [DEPHOSPHORYLATION PROTOCOL]: <br />
 +
Ω+AmyE 3’  S,P <br />
 +
Ω+AmyE 3’  II S,P <br />
 +
6 AraC+ Ω+AmyE 3’  E,X<br />
 +
8 AraC+ Ω+AmyE 3’  E,X<br />
 +
11 AraC+ Ω+AmyE 3’  E,X<br />
 +
12 AraC+ Ω+AmyE 3’  E,X<br /><br />•Ran a gel with A3 PCR [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (26) <br />
 +
 +
•It was the second time we didn’t obtain a band from A3’s PCR.
 +
 +
•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification. <br />
 +
 +
•Ran a gel woth E0040+B0014 X,P 4 to extract [GEL (27) ELECTROPHORESIS PROTOCOL] <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | Gen extraction protocol]]. <br />
 +
 +
•Did an A3 PCR [PCR PROTOCOL]. <br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
=SEPTEMBER=
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/b/b8/UnamgenomicsandsugarBitacora_28.jpg' height="300"/>
 +
<div class='captionrosa'>
 +
<p class='captionInside'>1.A3 PCR 1. <br />
 +
2.A3 PCR 2. <br />
 +
3.E0040+B0014 X,P 1. <br />
 +
4.E0040+B0014 X,P 2. <br />
 +
5.Ω+AmyE 3’  dephosphorylated S,P. <br />
 +
6.Ω+AmyE 3’  dephosphorylated S,P II. <br />
 +
7.97 dephosphorylated S,P. <br />
 +
8.98 dephosphorylated S,P. <br />
 +
9.99 dephosphorylated S,P. <br />
 +
10-13. Arac+ Ω+AmyE 3’  dephosphorylated  E,X 12. <br />
 +
14. 1 kb Ladder. <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
 +
<h2>09/01/12</h2><br />
 +
•Ran a gel with [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] : (28) <br />
 +
 +
•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’  dephosphorylated S,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Ligation | LIGATION PROTOCOL]]. <br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<h2>09/03/12</h2><br />
 +
 +
•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
 +
 +
<h2>09/04/12</h2><br />
 +
•Ran gel with digestions: <br />
 +
B0079+GusA E,S 3,4,5<br />
 +
PVeg+XylR E,S 1,5,7,10,13,16 <br />
 +
[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
 +
•Purified A3 PCR 1,2. <br />
 +
 +
•Ran a gel of A3 PCR <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]]  and digested with X,P [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
 +
•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.
 +
 +
•Plated colonies that grew in a new plate. <br />
 +
 +
•Made liquid cultures of these <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
 +
•Repeated the ones that did not grow. <br />
 +
 +
•Digested E0040+B0014 4 with X,P[[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Digestion_protocol_.2820_.C2.B5l.29 | LIQUID CULTURE]]. <br />
 +
 +
•Ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_electrophoresis | GEL ELECTROPHORESIS PROTOCOL]] . <br />
 +
 +
•Extracted from gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+<br />
 +
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/c/c1/UnamgenomicsandsugarBitacora_29.jpg' height="300"/>
 +
<div class='captionaqua'>
 +
<p class='captionInside'>24 PBad/PXyl+E0040+B0014 tubes we extracted <br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/a/a4/UnamgenomicsandsugarBitacora_30.jpg' height="300"/>
 +
<div class='captionmoradoclaro'>
 +
<p class='captionInside'>1.E0040+B0014 X,P<br />
 +
2.E0040+B0014 X,P<br />
 +
3.PVeg+XylR E<br />
 +
4.B0079+GusA S<br />
 +
5.1 kb plus ladder<br />
 +
Extracted band from 1. and 2
 +
<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/7/7d/UnamgenomicsandsugarBitacora_31.jpg' height="300"/>
 +
<div class='captionmorado'>
 +
<p class='captionInside'>Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S
 +
<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>09/05/12</h2><br />
 +
•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids  (29) [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL] and ran a gel [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
 +
•Extracted plasmid from 2 tubes of E0040+B0014 [PLASMID  EXTRACTION (“soft” lysis) PROTOCOL]. <br />
 +
 +
•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (30)PROTOCOL]: <br />
 +
1.E0040+B0014 X,P<br />
 +
2.E0040+B0014 X,P<br />
 +
3.PVeg+XylR E<br />
 +
4.B0079+GusA S<br />
 +
5.1 kb plus ladder<br />
 +
 +
•Extracted band from 1. and 2. [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Gel_extraction_protocol | LIQUID CULTURE]]. <br />
 +
 +
•Purified A3 PCR from 2 0.6ml tubes 1 and 2. <br />
 +
 +
•1 colony grew from ligation pVeg+E0040+B0014. <br />
 +
 +
•We streaked this in another plate and did liquid cultures <br /> [[Team:UNAM_Genomics_Mexico/Notebook/Protocols#Liquid_culture | LIQUID CULTURE]]. <br />
 +
 +
•To do: <br />
 +
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and  Ω+AmyE 3’  S,P dephosphorylated+ K143001 X,P. <br />
 +
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31) <br />
 +
 +
<html>
 +
<div class='thumbnailWrapper'>
 +
<ul>
 +
<li>
 +
<img src='https://static.igem.org/mediawiki/2012/2/26/UnamgenomicsandsugarBitacora_32.jpg' height="300"/>
 +
<div class='captionazul'>
 +
<p class='captionInside'>•A3 PCR and gel A3 PCR X,P 
 +
<br />
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
 +
<br /><br />
 +
</html>
 +
 +
<h2>09/06/12</h2><br />
 +
 +
•A3 PCR and gel A3 PCR X,P 1,2 [PCR PROTOCOL] [GEL (32)ELECTROPHORESIS PROTOCOL]. <br />
 +
 +
 +
<h2>09/14/12</h2><br />
 +
 +
•Digested  Ω+AmyE 3’  11,13,18, and 22 with E,X. <br />
 +
•Ligate: <br />
 +
PVeg S,P dephosphorylated+XylR X,P. <br />
 +
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P. <br />
 +
pSB13C3 E,P dephosphorylated + A3 PCR E,P. <br />
 +
pSB13C3 E,P dephosphorylated + GusA PCR E,P. <br />
 +
pSB13C3 E,P dephosphorylated + XylR E,P. <br />
 +
pSB13C3 E,P dephosphorylated + pVeg E,P. <br />
 +
pSB13C3 E,P dephosphorylated + pBad pXyl E,P. <br />
 +
pSB13C3 E,P dephosphorylated +  Ω PCR E,P. <br />
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
 +
 +
<br />
}}
}}

Revision as of 11:05, 26 September 2012


UNAM-Genomics_Mexico


Or Gate



2 JUNE


2.1 06/07/12
2.2 06/12/12
2.3 06/13/12
2.4 06/14/12
2.5 06/15/12
2.6 06/18/12
2.7 06/19/12
2.8 06/22/12
2.9 06/25/12
2.10 06/26/12
2.11 06/27/12
2.12 06/29/12

3 JULY


3.1 07/03/12
3.2 07/04/12
3.3 07/06/12
3.4 07/07/12
3.5 07/08/12
3.6 07/09/12
3.7 07/10/12
3.8 07/11/12
3.9 07/12/12
3.10 07/13/12
3.11 07/14/12
3.12 07/16/12
3.13 07/17/12
3.14 07/18/12
3.15 07/19/12
3.16 07/20/12
3.17 07/23/12
3.18 07/24/12
3.19 07/25/12
3.20 07/26/12
3.21 07/27/12
3.22 07/30/12
3.23 07/31/12

4 AUGUST


4.1 08/01/12
4.2 08/02/12
4.3 08/03/12
4.4 08/06/12
4.5 08/08/12
4.6 08/09/12
4.7 08/10/12
4.8 08/13/12
4.9 08/16/12
4.10 08/20/12
4.11 08/21/12
4.12 08/22/12
4.13 08/23/12
4.14 08/24/12
4.15 08/25/12
4.16 08/28/12
4.17 08/29/12
4.18 08/30/12
4.19 08/31/12

5 SEPTEMBER


5.1 09/01/12
5.2 09/03/12
5.3 09/04/12
5.4 09/05/12
5.5 09/06/12
5.6 09/14/12

UnmagenomicsBacteria sugar.png

On hover the images to see descriptions


JUNE



  • 1. 1 kb ladder
    2.E1010



06/07/12


PY BROTH 10g salt/L
PSB2K3 kanamicin
We transformed RFP E1010.
plate 1 18F
plate 2 17E
Stock 50 mg/mL
E.coli 1/1000
TRANSFORMATION PROTOCOL.
We made liquid cultures with colonies LIQUID CULTURE.
We extracted plasmids PLASMID EXTRACTION PROTOCOL.
We ran a gel to check the extraction GEL ELECTROPHORESIS PROTOCOL.




06/12/12


We made glycerols of the bacteria
GLYCEROL PROTOCOL .
We transformed plasmid pHp45 TRANSFORMATION PROTOCOL.

  • 1. 1 kb ladder
    2.E1010



06/13/12


We ran gel and extracted from gel LIQUID CULTURE.
We made liquid cultures LIQUID CULTURE and lysed [LYSIS PROTOCOL].














  • 1. 1 kb ladder
    We extracted from gel



06/14/12


We extracted from gel LIQUID CULTURE.
We made glycerols of pHp45 Ω GLYCEROL PROTOCOL .
We transformed PSB2K3 Km (Kanamycin) 5C BBa_J04450 PSB4A5 Am (ampicillin) 1I BBa_J04450 AraC BBa_C0080
2012 14L plate 1 pSB2K3 Km+
2011 14L plate 1 pSB2K3 Km+
2012 14L plate 1 pSB2K3 Km+
TRANSFORMATION PROTOCOL.





  • 1. 1 kb ladder
    pHp45 Ω with E/P
    We extracted from gel



06/15/12


> Digested pHp45 Ω with E/P LIQUID CULTURE.
> We extracted plasmid with kit.
> Ran a gel GEL ELECTROPHORESIS PROTOCOL . (5)
>Extracted from gel LIQUID CULTURE.

ARAC
>From the overnight plate we prepared liquid culture LIQUID CULTURE.
>We left them incubating overnight.
>We left a plate (LB Km DH5α C0080 and a control).





06/18/12


>Plasmid extraction AraC with kit.
>C0080-psB2K3 915 bp
>Streaked 4 LB Km 30 plates DH5α psB2K3.
>It seemed as though there were two different colonies (white) and there had been a mutation. Normally they are red.
>Ran gel with Spr/Strr (eppendorf -20ºC) GEL ELECTROPHORESIS PROTOCOL.
>Made glycerols from one tube of DH5α C0080 Km 50 -> 04.
>Made glycerols from one tube of DH5α PSB4A5 Amp 100 -> 05 GLYCEROL PROTOCOL.
>Digested C0080 X,S.
>Digested B0014 E,P LIQUID CULTURE.


  • 1. 1 kb ladder
    Digested B0014 with E, P and with E,X





06/19/12


>Digested B0014 with E, P and with E,X. LIQUID CULTURE.
>Ran gels with yesterday’s digestions GEL ELECTROPHORESIS PROTOCOL .
2 B0014 E,X and 3 B0014 E,P were discarded because of problems in the preparation.
C0080 is in the first lane.
>Extracted from gel C0080 X,S LIQUID CULTURE.
>Made glycerols from 4 plates LB Km DH5α pSB2K3 GLYCEROL PROTOCOL .
>Take out AmyE 5’ from distribution [DNA KIT PLATE INSTRUCTIONS].
AmyE 5’ 18K plate 3 2010, 2011, 2012
AmyE 3’ 18M plate 3 2010, 2011, 2012
>Digested B0014 with E,X and E,P again LIQUID CULTURE.






06/22/12


>Ran gels with digestions B0014 with E,X and E,P GEL ELECTROPHORESIS PROTOCOL.

>Dephosphated B0014 E,X and B0014 E,P [DESPHOPHORYLATION PROTOCOL].










06/25/12


>Took LasR BBa_B0079 plate 1 2010,2011,2012 psB1A2 Amp+ from distribution [DNA KIT PLATE INSTRUCTIONS].

06/26/12


>Ran gel with psB2K3 and psB4A5 GEL ELECTROPHORESIS PROTOCOL .

06/27/12


>Transformed with plasmid B0079 1576 bp psB1A2 12A TRANSFORMATION PROTOCOL.
LasR B0079 plasmid Amp+ 12A plate 1 2010,2011
AmyE 5’ BBa_K143001 Km+ 18K plate 3 2010 and 16M, 18K 2011
AmyE 3’ BBa_K143002 Km+ 18M plate 3 2010 and 16O, 18M 2011
AmyE 5’ grew 2 colonies.

06/29/12


>We did a DH5α K143001 Km30 Amp 100 glycerol GLYCEROL PROTOCOL 07.
>We plated twice DH5α K143002 Km 30 Amp 100 + control and twice DH5α B0079 Amp 100 + control
These were both plated in 2 plates each.
They were left overnight since they had not grown by 7:00 pm so the next day glycerols were made.
>Liquid cultures LIQUID CULTURE.

2 tubes DH5α K143001 Km30 Amp 100
2 tubes DH5α K143002 Km30 Amp 100
2 tubes DH5α B0079 Amp 100
1 tube LB Km 30 Amp 100 control
1 tube LB Amp 100 control
>From the 6 tubes we extracted plasmid from kit.


JULY

07/02/12
>Digestions LIQUID CULTURE.
B0079 digestion with S,P
K143001 with S,P
K143002 with S,P
>PCR’s
•AraC
•Cassete ΩSpr/Strr
PCR PROTOCOL

  • After Cassete ΩSpr/Strr PCR we ran a gel



  • B0079 digestion with S,P
    K143001 with S,P
    K143002 with S,P



07/03/12


>After Cassete ΩSpr/Strr PCR we ran a gel GEL ELECTROPHORESIS PROTOCOL . (8)
>Ran gel with digestions from yesterday. (9)
>Did band extractions LIQUID CULTURE.
>Stored at -20ºC.
>Did PCR to Cassete ΩSpr/Strr to add RBS and prefix/suffix [PCR PROTOCOL].
>Left digesting with E,S LIQUID CULTURE.






  • We dephosphated B0079 S,P, K143001 S,P, K143002 E,X



07/04/12


>We dephosphated B0079 S,P, K143001 S,P, K143002 E,X (9.1) [DESPHOPHORYLATION PROTOCOL].
>Ligated Cassete ΩSpr/Strr +K143002 dephosphated(digested with E,X and E,S) LIGATION PROTOCOL].
>Transformed ligation and left overnight plated TRANSFORMATION PROTOCOL.
>Extracted plasmid PBBR1 GusA in 2 1.5 mL eppendorfs [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
>Digested with PstI LIQUID CULTURE.






07/06/12


4 LB Km30 Spec60 DH5α ΩSpr/Strr +K143002 still have not grown.
Ran a gel with yesterday’s digestions to chek if they were done properly (10)
GEL ELECTROPHORESIS PROTOCOL .
Ran another gel with the rest of the samples.
Extracted GusA fragment
LIQUID CULTURE.
Repeated ligation ΩSpr/Strr +K143002 E,X dephosphated LIGATION PROTOCOL.
Repeated ΩSpr/Strr PCR.


07/07/12


Transformed DH5α ΩSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002 .


07/08/12


Transformed DH5α ΩSpr/Strr +K143002 ligation LIGATION PROTOCOL.
Plated LB Km30 Spec 60 DH5α ΩSpr/Strr +K143002.



  • We dephosphated B0079 S,P, K143001 S,P, K143002 E,X



  • 1 PCR omega P
    2 PCR omega I
    3 PCR AraC P
    4 PCR AraC I
    5 ladder 1 Kb



07/09/12


From yesterday’s transformations only one colony grew.
From the previous transformation only 2 colonies grew.
These 3 were streaked in 3 plates:
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 1.
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 2.
DH5α ΩSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km 30 Sp 100 control.
Did liquid cultures in 3 tubes:
ΩSpr/Strr +K143002 LB Km 30 Sp 100 1.
ΩSpr/Strr +K143002 LB Km 30 Sp 100 2.
ΩSpr/Strr +K143002 LB Km 30 Sp 100 3.
LB Km30 Sp 100 control.
LIQUID CULTURE.

Ran a gel with: (11)
GusA P
PBBR1 GusA
Ω E PCR P
Ω PCR P
Ω PCR I
Ω E
Ω PCR I
GEL ELECTROPHORESIS PROTOCOL
Did GusA primers dissolution for PCR.
GusA PCR
[PCR PROTOCOL]
Gel Extraction by kit of lanes 3 and 5 LIQUID CULTURE.

PCR omega P, PCR omega I, PCR AraC P, PCR AraC I
Add 10 μl of each primer (LW and UP).
Add 3 μl of plasmid (P).
Add 30.4 μl buffer.
Add 5 μl Mg.
Add 8 μl DNTp’s.
Add 42.6μl H2O miliQ.
Add 1 μl RTTG polymerase.
Centrifuge (spin) 8 secs.
Add vegetable oil till the eppendorf is full.
Place eppendorf 1 mL in thermocycler.
Run PCR with program “BERNA”.

  • GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S



07/10/12


•GusA digestions with X,P; Ω+AmyE 3’ with E,X; AraC PCR with E,S (2 times) DIGESTIONS. (11.2)











07/11/12


•GusA PCR digestions with E,S; Ω+AmyE 3’ with E,X LIQUID CULTURE.
•T.V. AraC PCR+ Ω+AmyE 3’ E,X dephosphated ligation LIGATION PROTOCOL.

  • 1. ladder.
    2. GusA PCR E,S.
    3. Ω+AmyE 3’ with E,X.



07/12/12


•Ran a gel with yesterday’s digestions: (12)

GEL ELECTROPHORESIS PROTOCOL .

•Transformed AraC PCR+ Ω+AmyE 3’ ligation in two tubes TRANSFORMATION PROTOCOL.

•From pfrc54 liquid cultures (A3) that were grown overnight we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Made glycerols from 2 LB Km 30 Sp100 DH5α Ω+AmyE 3’ plates and from 1 LB Amp100 pfrc54 (A3) plates with control GLYCEROL PROTOCOL .
•Ligated GusA PCR with B0014 E,X desphophorylated LIGATION PROTOCOL.

•Digested Pfrc54 (A3) with S,P LIQUID CULTURE.

•Desphophorylated Ω+AmyE 3’ E,X [DESPHOPHORYLATION PROTOCOL].







  • •Ran gel with pfrc54 S,P



07/13/12



•Ran gel with pfrc54 S,P GEL ELECTROPHORESIS PROTOCOL . (13)
•Transformation of GusA PCR + B0014 ligation TRANSFORMATION PROTOCOL.
•Transformed with GFP E0040 psBIA2.
•Liquid Cultures with ligation: AraC+ Ω+AmyE 3’ for plasmid extraction at night TRANSFORMATION PROTOCOL.
•B. Subtitils competent cells [B.Subtilis group protocol].








07/14/12



•From 24 AraC+ Ω+AmyE 3’ LB Km 30 Sp 100 DH5α tubes:
•Extracted pellets
•Extracted plasmids [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Ran gel GEL ELECTROPHORESIS PROTOCOL .

•From transformed DH5α km 30:
GusA+B0014 DH5α Km50 24 pellets
E0040 DH5α LB Amp100
From these two we:
•Did liquid cultures LIQUID CULTURE.
•Extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•From the GusA+E0014 transformation we took 24 colonies, did liquid cultures and extracted plasmid LIQUID CULTURE.
•Ran gel with this transformation GEL ELECTROPHORESIS PROTOCOL .



07/16/12



•Digested E,P AraC+ Ω+AmyE 3’
•Ω+AmyE 3’ E,P LIQUID CULTURE.


  • Digested E,P AraC+ Ω+AmyE 3’
    Ω+AmyE 3’ E,P



07/17/12



•Ran gel with yesterday’s digestions LIQUID CULTURE. (14)
•The gel we ran didn’t work, probably because the agarose was not prepared correctly.
•Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S LIQUID CULTURE.











  • •Digested 12, 15, 18, 21 with AraC+ Ω+AmyE 3 with E and also with P’; B0014 with E,X; E0040 with X,S




07/18/12



•From yesterday’s digestions we ran a gel GEL ELECTROPHORESIS PROTOCOL . (15)

•We made liquid cultures of AraC+ Ω+AmyE 3’ 12, 15, 21 LIQUID CULTURE.

•We ligated AraC PCR E,S dephosphated with + Ω+AmyE 3’ LIGATION PROTOCOL.

•We digested 1 + Ω+AmyE 3’ E,P LIQUID CULTURE.

07/19/12



•We ran We digested 1 + Ω+AmyE 3’ E,P with Miguel’s enzymes and with Abiel’s enzymes LIQUID CULTURE.

•We transformed with ligations AraC+ Ω+AmyE 3’ E and + Ω+AmyE 3’ E TRANSFORMATION PROTOCOL.

•Extracted plasmids from AraC+ Ω+AmyE 3’ 12,15,21 [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].

•Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P LIQUID CULTURE.



  • Digested E0040 X,S 2,3,4,5,6; ligations AraC+ Ω+AmyE 3’ E 12,15,21; ligations AraC+ Ω+AmyE 3’ P



07/20/12



•Ran gel with yesterday’s digestions GEL ELECTROPHORESIS PROTOCOL . (16)
•Transformed GusA+B0014 in two tubes TRANSFORMATION PROTOCOL.
•Made liquid cultures from AraC+ Ω+AmyE 3’s transformation LB Km30 Sp 100 and control LB Km 30 Sp100 LIQUID CULTURE.






07/23/12



•From 24 GusA+B0014 tubes (-) we didn’t do anything.
•From 4 AraC+ Ω+AmyE 3’ we extracted plasmid [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Digested from the 4 AraC+ Ω+AmyE 3’ E,X; X; P and GusA PCR with X,P LIQUID CULTURE.

07/24/12



•Digested B0014 E with X B0014 X with E
•Digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P LIQUID CULTURE.

07/25/12



•Ran gel of digested AraC+ Ω+AmyE 3’ 1,2,3,4 with E,P LIQUID CULTURE.
•Joined B0014 E with X B0014 X with E digestions.
•Made liquid cultures from 2 days old plated GusA+B0014 and extracted plasmid LIQUID CULTURE [PLASMID EXTRACTION (“soft” lysis) PROTOCOL]. Transformed TRANSFORMATION PROTOCOL.
•From LasR DH5α make liquid cultures and plate LIQUID CULTURE.
•Digested Ω+AmyE 3’ with X,P; E,S; C0080 X,P; E,S; S,P LIQUID CULTURE.
•Ligated Gus PCR X,P with B0079 S,P dephosphorylated LIGATION PROTOCOL.


  • 1.Ω+AmyE 3’ X,P
    2. Ω+AmyE 3’ E,S
    3.C0080 X,P
    4.C0080 E,S
    5.C0080 S,P
    6.ladder



07/26/12


•From C0179 (LasR) transformation which grew colonies, we made two liquid cultures: DH5α C0179 LB Cb 100 (two cultures) and a control without bacteria LIQUID CULTURE.

•We extracted plasmid from 8 C0179 tubes, from 2 tubes of C0179 (Miguel’s transformation), Puc, B.S. control and PSB4AE [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].

•Due to problems with the way we did the transformations of ligations we repeated them:
GusAPCR X,P+ B0079 S,P dephosphorylated

GusAPCR X,P+ B0079 S,P dephosphorylated (-)

GusAPCR X,P+ A3 S,P dephosphorylated

GusAPCR X,P+ A3 S,P dephosphorylated (-)
TRANSFORMATION PROTOCOL.

•Did the following digestions LIQUID CULTURE.
•AraC+ Ω+AmyE 3’ S,P 1,2,3,4
•K143002 X,P
•AraC+ Ω S,P
•C0179 X,S


07/27/12



Ligated AraC+ Ω S,P dephosphorylated + K143002 X,P LIGATION PROTOCOL.

07/30/12



•Transformed with ligations:
•AraC+ Ω dephosphprylated +K143002 X,P
•GusAPCR X,P+ A3 S,P dephosphorylated
•GusAPCR X,P+ B0079 S,P dephosphorylated
•Transformed the following sythesis:
•91996 Pveg 140 bp
•91997 ArsR-CzrA_promoter 1 194 bp
•91998 ArsR-CzrA_promoter 2 221 bp
•91999 ArsR-CzrA_promoter 3 213 bp
•92000 pBad-pXyl 387 bp
•92001 XylR 1117pb
•92002 CI_pro_(NAND_INHIBITOR) 774
•These synthesis come in pJ209 and supposedly come with Chloramphenicol+ and Gentamicin+ resistance, but we discovered that only Chloramphenicol worked. TRANSFORMATION PROTOCOL.

•Make liquid cultures of the following transformations for tomorrow LIQUID CULTURE:
•AraC+ Ω+K143002
•GusA+A3
•GusA+B0079
•Synthesis

07/31/12



Extracted plasmids from yesterday’s cultures and from K143002 cultures that were in the other lab [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].


AUGUST

08/01/12



Digested XylR 01 X,P; pBAD-pXyl X,P; Pveg 96 S,P LIQUID CULTURE.


  • 1 PCR GusA I
    2 PCR GusA I
    3 PCR GusA P
    4 PCR GusA P
    5 ladder 1 Kb



08/02/12



•Extracted plasmids from liquid cultures[PLASMID EXTRACTION (“soft” lysis) PROTOCOL]:
AraC+ Ω+K143002
•GusA+A3
•GusA+BBR1

PCR GusA, GusA I, PCR GusA P, PCR GusA I

•Add 10 μl of each primer (LW and UP).
•Add 3 μl of plasmid (P).
•Add 30.4 μl buffer.
•Add 5 μl Mg.
•Add 8 μl DNTp’s.
•Add 42.6μl H2O miliQ.
•Add 1 μl RTTG polymerase.
•Centrifuge (spin) 8 secs.
•Add mineral oil till the eppendorf is full.
•Place eppendorf 1 mL in thermocycler.
•Run PCR with program “BERNA”.



  • 1.GusA PCR 1
    2.GusA PCR 2
    3.A3+GusA 1
    4.A3+GusA 2
    5.A3+GusA 3
    6.P GusA
    7.P GusA 2
    8.Ladder
    9.01
    10.06
    11.96





08/03/12



• Ran a gel with: (18)

GEL ELECTROPHORESIS PROTOCOL .
•Ran another gel to extract with:
1.GusA PCR 1
2.GusA PCR 2
3.00
4.01
GEL ELECTROPHORESIS PROTOCOL .
•Did the following digestions LIQUID CULTURE:

• Ω+K143002 E,X; A3+GusA E,S; GusAPCR X,P

•Extracted GusAPCR X,P digestion LIQUID CULTURE.

•Left the following ligation: AraC+ Ω+K143002 LIGATION PROTOCOL.




  • 1.A3+GusA 2
    2.A3+GusA 3
    3. Ω+AmyE 3’ 2
    4. Ω+AmyE 3’ 3
    5.XylR X,P
    6.pBad-pXyl X,P
    7.GusA PCR X,P




08/06/12



•Made liquid cultures of Ω+K143002 (2 cultures+1 control) and AraC+ Ω+K143002 (2 cultures+1 control) LIQUID CULTURE.
•Ligated GusAPCR X,P+B0079 S,P dephosphorylated LIGATION PROTOCOL.
•Ran a Gel with: (19)









  • Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P



08/08/12


BBa_B0040 6I psB1A2 Amp+ plate 1
Digested 14 tubes of AraC+ Ω+AmyE 3’ E,P LIQUID CULTURE. (19.1)








08/09/12


•From yesterday’s PCR’s :
• E0040 plasmid 1
E0040 plasmid 2
E0040 digested 1
E0040 digested 2
We purified with PCR kit

•Ran a gel GEL ELECTROPHORESIS PROTOCOL .
•Made 14 PCR’s from AraC+ Ω+AmyE 3’.
•From B0079+GusA ligation and B0040 transformation: •Grew colonies.
•Made liquid cultures LIQUID CULTURE.
•Streaked these in a new plate.
•Diluted plasmid E0080 2 1/50.


08/10/12


•Yesterday’s gel did not come out as expected so we repeated the PCR.



  • 1.AraC+ Ω+AmyE 3’
    2-14.AraC+ Ω+AmyE 3’
    15.AraC+ Ω 1
    16.AraC+ Ω 1
    17.AraC+ Ω 1
    18.PCR 1 GFP
    19.PCR GFP




08/13/12


•After running a gel with 14 PCR’s AraC+ Ω+AmyE 3’ and it did not come out as expected, we ran a gel with the ligation AraC+ Ω+AmyE 3’ to select candidate (that weight the same as the ligation) GEL ELECTROPHORESIS PROTOCOL : (20)

•Extracted pasmids form liquid cultures: B0049, B0079+GusA [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].

•AraC+ Ω+AmyE 3’ undigested didn’t run in the gel as expected, it ran similarly to AraC+ Ω

• We repeated the ligation AraC+ Ω S,P dephosphorylated+K143002 X,P LIGATION PROTOCOL.

• GFP PCR gel came out with two bands one is GFP and the other one are the primers, even after purifying by kit. We joined 2 GFP PCR tubes.

• We ran a gel to extract.




  • 1.6 AraC+ Ω+AmyE 3’ E with P.

    2.8 AraC+ Ω+AmyE 3’ E with P.
    3.11 AraC+ Ω+AmyE 3’ E with P.
    4.12 AraC+ Ω+AmyE 3’ E with P.
    5.6 AraC+ Ω+AmyE 3’ with E,X.
    6.8 AraC+ Ω+AmyE 3’ with E,X.
    7.11 AraC+ Ω+AmyE 3’ with E,X.
    8.12 AraC+ Ω+AmyE 3’ with E,X.
    9.00 pBad/pXyl with S,P.
    10.Ladder.
    11.E0040 PCR E,S to extract the correct band.




08/16/12


•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (22)PROTOCOL]:

•Digested:
ArSR-CzrA 97 with S,P
ArSR-CzrA 98 with S,P
ArSR-CzrA 99 with S,P
E0040 PCR with E,S
Digestion Protocol.






08/20/12


•Ligated B0079 S,P dephosphorylated + GusA PCR X,P and pVeg S,P dephosphorylated + XylR X,P LIGATION PROTOCOL.


08/21/12


•Joined both .6 ml tubes of B0014 EX and B0014 XE in one and dephosphorylated [DEPHOSPHORYLATION PROTOCOL].
•Extracted plasmid from the 24 tubes of of ligation K143001+pBad/pXylR [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].
•Digested Ω+AmyE 3’ with E,X and + Ω+AmyE 3’ II with E,X (obtained these two from glycerols stored at -80ºC LIQUID CULTURE.


  • Digested K143001+PBad, pXyl with E,S



  • 1.K143001+PBad, pXyl E,S 10
    2.K143001+PBad, pXyl E,S 11
    3.K143001+PBad, pXyl E,S 12
    4.A3 PCR
    5.A3 PCR
    6.E0040 PCR E,S
    7.B0014 E,X dephosphorylated
    8. Ω+AmyE 3’ E,X
    9. Ω+AmyE 3’ E,X II
    10.Ladder



08/22/12


•We did 2 PCR’s for A3 [PCR PROTOCOL].

•Digested K143001+PBad, pXyl with E,S
LIQUID CULTURE. (22.1)

GEL ELECTROPHORESIS PROTOCOL .
•Did band extraction of:
1. K143001+PBad, pXyl E,S 10
2.K143001+PBad, pXyl E,S 11
3.K143001+PBad, pXyl E,S 12

LIQUID CULTURE.
•Made liquid cultures from E1010+Boo14 that the other lab gave us and did 2 plates for glycerol Km+ LIQUID CULTURE
GLYCEROL PROTOCOL .

•Did 2 plates for glycerol of both K143001+pBad/pXyl 10y11 Km+ and AraC+ Ω+AmyE 3’ 6,8,11,12 Km+ and Spectinomycin+ GLYCEROL PROTOCOL .

•Transformed ligations:
B0079+GusA Amp+
Pveg+XylR Chloramphenicol+ TRANSFORMATION PROTOCOL..


  • 1. Ω+AmyE 3’ E,X 6.2
    2.Ω+AmyE 3’ II E,X
    3.97 ArsR-CzrA
    4.98 ArsR-CzrA
    5.99 ArsR-CzrA



08/23/12


•The negative controls were contaminated. We didn’t manipulate the ones that had grown because of this.
•Ran gel with GEL ELECTROPHORESIS PROTOCOL : (23.01)
Digestion Protocol.


08/24/12


From plate with ligation E0040+B0014, ligation K143001+pBad,pXyl 10 and 11, and ligation AraC+ Ω+AmyE 3’ 6 and 8 we streaked 2 plates for glycerol of each ligation.
Repeated ligation B0079+GusA.

Ligated E0040 PCR E,S+B0014 E,X dephospohylated LIGATION PROTOCOL.

Made 2 new plates from Pveg+XylR ligation and liquid cultures LIQUID CULTURE.


08/25/12


Extracted pellet from 16 tubes of pVeg+XylR that were left overnight [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].

Ligated B0079+GusA and B0040+B0014 LIGATION PROTOCOL.


08/28/12


Made liquid cultures from transformations tht were left overnight -R0079+GusA
-R0079+GusA (-)
-E0040+B0014
-E0040+B0014 (-)
LIQUID CULTURE.

  • 1. A3 PCR 1.
    2. A3 PCR 2.
    3. Ω+AmyE 3’ S,P *.
    4. Ω+AmyE 3’ S,P .
    5. Pveg+XylR E,S 2.
    6. Pveg+XylR E,S 3.
    7. Pveg+XylR E,S 4.



08/29/12


Digested Pveg+XylR 2,3,4 with E,S.

Digested Ω+AmyE 3’ with S,P DIGESTION.

Ran a PCR with A3 PCR (2 .6 ml tubes) [PCR PROTOCOL].

Ran a gel with
GEL ELECTROPHORESIS PROTOCOL : (23.01)








  • 1-23. E0040 + B0014 colonies
    24-29. B0079+GusA 1
    30. B0079



08/30/12


•Ran a gel GEL ELECTROPHORESIS PROTOCOL : (24)


•Digested PVeg+XylR 1,5,7,10,13,16 with E,S; E0040+B0014 3,4,5,10,11,19,21,22 with X,P; B0079+GusA 3,4,5 E,S LIQUID CULTURE.









  • 1-3B0079+GusA E,S 3.
    4-9. PVeg+XylR E,S 1.
    10-17. E0040+B0014 X,P 3.
    18. 1 kb ladder.



  • 1.A3 PCR.



  • E0040+B0014 X,P 4 to extract



08/31/12


•Ran a gel GEL ELECTROPHORESIS PROTOCOL : (25)


•Dephosphorylated [DEPHOSPHORYLATION PROTOCOL]:
Ω+AmyE 3’ S,P
Ω+AmyE 3’ II S,P
6 AraC+ Ω+AmyE 3’ E,X
8 AraC+ Ω+AmyE 3’ E,X
11 AraC+ Ω+AmyE 3’ E,X
12 AraC+ Ω+AmyE 3’ E,X

•Ran a gel with A3 PCR GEL ELECTROPHORESIS PROTOCOL : (26)

•It was the second time we didn’t obtain a band from A3’s PCR.

•Probably because it had been missing the buffer 2’s ethanol (“blue lid”) from the PCR purification.

•Ran a gel woth E0040+B0014 X,P 4 to extract [GEL (27) ELECTROPHORESIS PROTOCOL]
Gen extraction protocol.

•Did an A3 PCR [PCR PROTOCOL].






SEPTEMBER

  • 1.A3 PCR 1.
    2.A3 PCR 2.
    3.E0040+B0014 X,P 1.
    4.E0040+B0014 X,P 2.
    5.Ω+AmyE 3’ dephosphorylated S,P.
    6.Ω+AmyE 3’ dephosphorylated S,P II.
    7.97 dephosphorylated S,P.
    8.98 dephosphorylated S,P.
    9.99 dephosphorylated S,P.
    10-13. Arac+ Ω+AmyE 3’ dephosphorylated E,X 12.
    14. 1 kb Ladder.




09/01/12


•Ran a gel with GEL ELECTROPHORESIS PROTOCOL : (28)

•Ligated E0040+B0014 X,P 2 +promoter S,P dephosphorylated; K143002 X,P+ Ω+AmyE 3’ dephosphorylated S,P LIGATION PROTOCOL.





09/03/12


•Digested B0079+GusA E,S 3,4,5; PVeg+XylR E,S 1,5,7,10,13,16 LIQUID CULTURE.


09/04/12


•Ran gel with digestions:
B0079+GusA E,S 3,4,5
PVeg+XylR E,S 1,5,7,10,13,16
GEL ELECTROPHORESIS PROTOCOL .

•Purified A3 PCR 1,2.

•Ran a gel of A3 PCR
GEL ELECTROPHORESIS PROTOCOL and digested with X,P LIQUID CULTURE.

•From ligations: PVeg+E0040+B0014, PBad/PXyl+E0040+B0014, B0079+E0040+B0014 only colonies grew in PBad/PXyl+E0040+B0014.

•Plated colonies that grew in a new plate.

•Made liquid cultures of these
LIQUID CULTURE.

•Repeated the ones that did not grow.

•Digested E0040+B0014 4 with X,P LIQUID CULTURE.

•Ran a gel GEL ELECTROPHORESIS PROTOCOL .

•Extracted from gel LIQUID CULTURE.
-BBa_J23101 18E plate 1 plasmid BBa_J61002_2948 bp Amp+


  • 24 PBad/PXyl+E0040+B0014 tubes we extracted



  • 1.E0040+B0014 X,P
    2.E0040+B0014 X,P
    3.PVeg+XylR E
    4.B0079+GusA S
    5.1 kb plus ladder
    Extracted band from 1. and 2



  • Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S



09/05/12


•From the 24 PBad/PXyl+E0040+B0014 tubes we extracted plasmids (29) [PLASMID EXTRACTION (“soft” lysis) PROTOCOL] and ran a gel LIQUID CULTURE.

•Extracted plasmid from 2 tubes of E0040+B0014 [PLASMID EXTRACTION (“soft” lysis) PROTOCOL].

•Ran a gel with yesterday’s digestions [GEL ELECTROPHORESIS (30)PROTOCOL]:
1.E0040+B0014 X,P
2.E0040+B0014 X,P
3.PVeg+XylR E
4.B0079+GusA S
5.1 kb plus ladder

•Extracted band from 1. and 2. LIQUID CULTURE.

•Purified A3 PCR from 2 0.6ml tubes 1 and 2.

•1 colony grew from ligation pVeg+E0040+B0014.

•We streaked this in another plate and did liquid cultures
LIQUID CULTURE.

•To do:
•Ligate B0079 S,P dephosphorylated + E0040+B0014 X,P and Ω+AmyE 3’ S,P dephosphorylated+ K143001 X,P.
•Digested B0079+GusA with E,S 4,5; PVeg+XylR with E,S 10,13. (31)

  • •A3 PCR and gel A3 PCR X,P



09/06/12


•A3 PCR and gel A3 PCR X,P 1,2 [PCR PROTOCOL] [GEL (32)ELECTROPHORESIS PROTOCOL].


09/14/12


•Digested Ω+AmyE 3’ 11,13,18, and 22 with E,X.
•Ligate:
PVeg S,P dephosphorylated+XylR X,P.
PBad p Xyl S,P dephosphorylated + E0040+B0014 X,P.
pSB13C3 E,P dephosphorylated + A3 PCR E,P.
pSB13C3 E,P dephosphorylated + GusA PCR E,P.
pSB13C3 E,P dephosphorylated + XylR E,P.
pSB13C3 E,P dephosphorylated + pVeg E,P.
pSB13C3 E,P dephosphorylated + pBad pXyl E,P.
pSB13C3 E,P dephosphorylated + Ω PCR E,P.





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