Team:Groningen/Labwork/16 July 2013

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'''Mirjam'''
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<h2>Mirjam</h2>
<br/>Finally the colonies are growing on LB agar plates with ampicillin. The negative control does not show any colonies, the double negative does??, the positive control shows a lot of red colonies. On one of the four plates there are no colonies present, on two of them only a couple and on one a lot.
<br/>Finally the colonies are growing on LB agar plates with ampicillin. The negative control does not show any colonies, the double negative does??, the positive control shows a lot of red colonies. On one of the four plates there are no colonies present, on two of them only a couple and on one a lot.
The colonies are picked and placed in LB medium with ampicillin to grow further for miniprep.
The colonies are picked and placed in LB medium with ampicillin to grow further for miniprep.
 +
Colony PCR revealed no bands.
 +
 +
Mini prep analysis is done with the colonies of plate 1 and 3.
 +
<br/><a href="https://2013.igem.org/Team:Groningen/protocols/Digestion"><FONT COLOR="black"><b>restriction analysis</b></FONT></a> is done with EcoRV.
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 +
Picked colonies from the plate with the backbone of Munich (BBa_K823023) for overnight inoculation in LB medium with ampicillin. To obtain more sample of this backbone.
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'''Inne'''
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<h2>Inne</h2>
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<br> Did a PCR with 3 samples in duplo
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<br> Did a <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> with 3 samples in duplo
<table border="1">
<table border="1">
<tr>
<tr>
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<br> Ran the gel 1.5% agarose using the big slots, this posed a couple of problems:
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<br> Ran a <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>gel</b></FONT></a> 1.5% agarose using the big slots,  
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<br> The gel was to thick, the slots were still open but the electrophoresis machine needed to be filled up to the brim with 1x KBE buffer.
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<br> gel ran at 90 V for 45 minutes.
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<br> epje 3. (duplo) opened during the PCR so almost no solution was left.
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<br>gel didn't run nice, tried to cut out bands from 2 and 3.
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<br> added 60 uL gene ruler to the first slot, which is way to much.
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<br> did gel purification
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<br> gel ran ADD 90 V for 45 minutes, started foaming because of to much KBE.
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<br> Ran 0.8% agarose <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>gel</b></FONT></a> for 24 min on 100 V
 +
<br> Turned out that the bands were a bit higher then 1kb
 +
 
 +
<br>made new TBE buffer. 100ml TBE/900ml demiwater.
 +
 
 +
<br> made the inoculation of the <i>E.coli</i> with the BBa_k818000 biobrick for the Delft iGEM team.
 +
<br>Cells inoculated in 4 ml LB liquid medium with 8 uL of stock Ampicillin
 +
<br>Cells are to grow overnight in a 37 C shaker.
 +
 +
 
 +
<h2>Sander</h2>
 +
<br/>did a <a href="https://2013.igem.org/Team:Groningen/protocols/Digestion"><FONT COLOR="black"><b>restriction digestion</b></FONT></a> for Silk and Signal Sequences.
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<table border="1">
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<tr>
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<td>Sample</td>
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<td>componant</td>
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<td>volume (ul) </td>
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</tr>
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<tr>
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<td>silk1</td>
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<td>silk</td>
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<td>4</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI buffer</td>
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<td>2</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI enzyme</td>
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<td>0,5</td>
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</tr>
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<tr>
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<td>silk2</td>
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<td>silk</td>
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<td>4</td>
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</tr>
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<tr>
 +
<td></td>
 +
<td>BamHI buffer</td>
 +
<td>2</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI enzyme</td>
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<td>0,5</td>
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</tr>
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<tr>
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<td>silk3</td>
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<td>silk</td>
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<td>4</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI buffer</td>
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<td>2</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI enzyme</td>
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<td>0,5</td>
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</tr>
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<tr>
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<td>SS MotB</td>
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<td>MotB</td>
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<td>2</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI buffer</td>
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<td>2</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI enzyme</td>
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<td>0,5</td>
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</tr>
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<tr>
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<td>SS FliZ</td>
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<td>Fliz</td>
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<td>2</td>
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</tr>
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<tr>
 +
<td></td>
 +
<td>BamHI buffer</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>BamHI enzyme</td>
 +
<td>0,5</td>
 +
</tr>
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<tr>
 +
<td>SS EstA</td>
 +
<td>EstA</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>BamHI buffer</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>BamHI enzyme</td>
 +
<td>0,5</td>
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</tr>
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<tr>
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<td>SS LytB</td>
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<td>LytB</td>
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<td>2</td>
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</tr>
 +
<tr>
 +
<td></td>
 +
<td>BamHI buffer</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td></td>
 +
<td>BamHI enzyme</td>
 +
<td>0,5</td>
 +
</tr>
 +
</table>
 +
<br/>MilliQ was added to reach a 20 ul volume.   
 +
<br/>Incubation started at 11:00 for the signal sequences and 11:30 for the silk.
 +
<br/> at 15:38 the restriction digestion reaction was run over a 1,5 % agarose <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>gel</b></FONT></a> with 100 v for 22 min.
 +
<br/> no result on the gel for the sik. results as expected for signal sequence.   
 +
 
 +
<h2>Claudio and Mike</h2>
 +
 
 +
<br>The synthetic genes design goes on.
 +
<br>Today we focus on codon optimizing the sequences for <i>Bacillus Subtilis</i> taking into account the codon usage.
 +
 
 +
</div>
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 +
 
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</div>
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<div id="footer">
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  <p>iGEM 2013 Groningen</p>
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</div>
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'''Sander'''
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</body>
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<br/>did a Restriction digestion for Silk and Signal Sequences. incubation started at 11:00 for the signal sequences and 11:30 for the silk.
+
</html>

Latest revision as of 10:30, 30 July 2013

Mirjam


Finally the colonies are growing on LB agar plates with ampicillin. The negative control does not show any colonies, the double negative does??, the positive control shows a lot of red colonies. On one of the four plates there are no colonies present, on two of them only a couple and on one a lot. The colonies are picked and placed in LB medium with ampicillin to grow further for miniprep. Colony PCR revealed no bands. Mini prep analysis is done with the colonies of plate 1 and 3.
restriction analysis is done with EcoRV. Picked colonies from the plate with the backbone of Munich (BBa_K823023) for overnight inoculation in LB medium with ampicillin. To obtain more sample of this backbone.

Inne


Did a PCR with 3 samples in duplo
Sample Primer F Primer R Annealing temp.
1 without strep tag without strep tag 78 C
2 with strep tag without strep tag 78 C
3 without strep tag with strep tag 80 C
Protocol:
amount compound
28.2 uL MilliQ
1.5 uL 5% DMSO
1 uL each dNTP
10 uL Phusion GC buffer
2.5 uL Primer F
2.5 uL Primer R
1 uL Silk template BBa_16
0.3 uL Phusion polymerase

Ran a gel 1.5% agarose using the big slots,
gel ran at 90 V for 45 minutes.
gel didn't run nice, tried to cut out bands from 2 and 3.
did gel purification
Ran 0.8% agarose gel for 24 min on 100 V
Turned out that the bands were a bit higher then 1kb
made new TBE buffer. 100ml TBE/900ml demiwater.
made the inoculation of the E.coli with the BBa_k818000 biobrick for the Delft iGEM team.
Cells inoculated in 4 ml LB liquid medium with 8 uL of stock Ampicillin
Cells are to grow overnight in a 37 C shaker.

Sander


did a restriction digestion for Silk and Signal Sequences.
Sample componant volume (ul)
silk1 silk 4
BamHI buffer 2
BamHI enzyme 0,5
silk2 silk 4
BamHI buffer 2
BamHI enzyme 0,5
silk3 silk 4
BamHI buffer 2
BamHI enzyme 0,5
SS MotB MotB 2
BamHI buffer 2
BamHI enzyme 0,5
SS FliZ Fliz 2
BamHI buffer 2
BamHI enzyme 0,5
SS EstA EstA 2
BamHI buffer 2
BamHI enzyme 0,5
SS LytB LytB 2
BamHI buffer 2
BamHI enzyme 0,5

MilliQ was added to reach a 20 ul volume.
Incubation started at 11:00 for the signal sequences and 11:30 for the silk.
at 15:38 the restriction digestion reaction was run over a 1,5 % agarose gel with 100 v for 22 min.
no result on the gel for the sik. results as expected for signal sequence.

Claudio and Mike


The synthetic genes design goes on.
Today we focus on codon optimizing the sequences for Bacillus Subtilis taking into account the codon usage.

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