Team:Goettingen/notebook wetlab/biobricks

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                 <li><a href="/Team:Goettingen/notebook_wetlab">Wet lab</a>
                 <li><a href="/Team:Goettingen/notebook_wetlab">Wet lab</a>
                       <ul>
                       <ul>
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                       <li><a href="/Team:Goettingen/notebook_wetlab">BAIT Team</a></li>                       
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                       <li><a href="/Team:Goettingen/notebook_wetlab">PepTag Team</a></li>                       
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                       <li><a href="/Team:Goettingen/notebook_wetlab/prey_team">PREY Team</a></li>
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                       <li><a href="/Team:Goettingen/notebook_wetlab/biobricks">BioBrick Team</a></li>
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                       <li><a href="/Team:Goettingen/notebook_wetlab/biobricks">BIOBRICKS Team</a></li>
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                       <li><a href="/Team:Goettingen/notebook_wetlab/gfp">GFP Team</a></li>
                       </ul>
                       </ul>
                 </li>
                 </li>
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<div class="proRP" id="notebookWrap">
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<div class="proRP" id="BBWrap">
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         <h2>October</h2>
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         <br /><h2>July</h2><br />
-
<!--Template:
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<p>
-
                <div id="week2">
+
- Used BioBricks from library:<br />
-
                <h2>August 19th, week2</h2>
+
<img src="https://static.igem.org/mediawiki/2014/9/9b/Goettingen_TableBB1.png" width="600" />
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                <h4 class="collapsibleListTitle" id="lab_team1"><i>C. albicans & C. glabrata</i></h4>
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- Tick with a tip a hole in plate and resolve the pellet in 10µl sterile water, red color was visible, pipeting up and down, wait 5 minutes and put 10 µl into a PCR cup and freeze it<br />
-
                <p class="collapsibleListBody"></p>
+
- 1 µl is transformed into DH5α → plated on Cm or kan plates<br />
-
                <h4 class="collapsibleListTitle" id="lab_team2">Prey team</h4>
+
- Transformation worked, pick colonies for o/n in LB + Cm to isolate BB-plasmids<br />
-
                <p class="collapsibleListBody">It hasn't started yet.</p>
+
- Isolation of plasmids<br /><br />
-
                <h4 class="collapsibleListTitle" id="lab_team3">Biobrick team</h4>
+
<b>Agarose gel (1%):</b>
-
                <p class="collapsibleListBody">It hasn't started yet.</p>
+
<img src="https://static.igem.org/mediawiki/2014/7/77/Goettingen_FigureBB2.png" width="300" />
-
                </div>
+
<br />
 +
</p>
 +
       
 +
        <h2>August</h2><br />
 +
<p>
 +
<b>→After several tries to construct our BioBricks on the described way we changed to seamless cloning technique</b><br /><br />
 +
- PCRs for seamless cloning:<br />
 +
<img src="https://static.igem.org/mediawiki/2014/6/63/Goettingen_TableBB2.png" width="650" /><br />
 +
<b>Agarose gels (1%):</b><br />
 +
<img src="https://static.igem.org/mediawiki/2014/b/b9/Goettingen_FigureBB3.png" width="300" />
 +
<img src="https://static.igem.org/mediawiki/2014/c/c6/Goettingen_FigureBB4.png" width="300" />
 +
- Purification of the products and measurement of the concentration of each product.<br />
 +
- Amplification of Leu2, Trp1 and 2-micron with 2 sets of primers to create mutated genes that loose restriction sites:<br /><br />
-
备注: team1 is for Candida albicans and Candida glabrata; team2 is for A. fumigatus; team3 is for A. nidulans; team4 is for S. cerevisiae; team5 is for Toxoplasma gondii.
+
1. PCR to create for each gene 2 fragments that contain 15 bp overhangs to the other one.<br />
-
-->
+
2. Use fusion PCR approach to fuse genes with silent mution together.<br />
 +
3. Gel extract the rightly fused PCR product and use it in a normal PCR for amplification.<br />
 +
<img src="https://static.igem.org/mediawiki/2014/f/fb/Goettingen_FigureBB5.png" width="450" />
 +
- Purification and sequencing of this three samples <br />
 +
</p>
 +
<br /><h2>September</h2><br />
 +
<p>
 +
<b>- results sequencing:</b><br /><br />
 +
Trp1  AGGCCGCAGAATGTGCTCT<font color="#c33418"><b>G</b></font>GATTCCGATGCTGACTTGCT<br />
 +
Trp1*AGGCCGCAGAATGTGCTCT<font color="#c33418"><b>A</b></font>GATTCCGATGCTGACTTGCT<br />
 +
<br />
 +
Leu2 TGGTCCTAAATGGGGTAC<font color="#c33418"><b>C</b></font>GGTAGTGTTAGACCTGAACAA<br />
 +
Leu2*TGGTCCTAAATGGGGTAC<font color="#c33418"><b>A</b></font>GGTAGTGTTAGACCTGAACAA<br />
 +
<br />
 +
2-micron GAAGTTCCTATACTTTCTAG****AGAATAGGAACTTCGGAATAGGAACTTC<br />
 +
2-micron*GAAGTTCCTATACTTTCTAG<font color="#c33418"><b>CTAG</b></font>AGAATAGGAACTTCGGAATAGGAACTTC<br />
 +
<br />
 +
⇒ silent mutations delete the restriction sides<br />
 +
<br />
 +
- Prepare seamless mixture (see protocol and formula) with I – VI<br />
 +
- Transformation of the whole reaction sample into DH5α.<br />
 +
- Grown colonies on plates → pick several colonies and grow over night.<br />
 +
- Colony PCR with primer on backbone and primer in insert<br />
 +
- plasmid isolation of positives <br />
 +
<br />
 +
- Amplification of construct via PCR <br />
 +
<img src="https://static.igem.org/mediawiki/2014/f/f6/Goettingen_FigureBB1.png" width="250" />
 +
- Gel extraction & Purification & seamless cloning with A-F<br />
 +
- Transformation into DH5α<br />
 +
- Pick colonies and isolate plasmids from overnight culture<br />
 +
- colony PCR to verify construct<br />
 +
- restriction of the two different plasmids and the fusion product of 2-micron ligation over night of the 2-micron behind restriction site in plasmid backbones<br />
 +
- Transformation into DH5α.<br />
 +
- Pick several colonies into 4 mL LB +Cm for overnight cultures.<br />
 +
- Colony PCR and isolation of plasmids.<br />
 +
- Chose positives for further work and shipping to the registry<br /><br />
 +
</p>
 +
<div><a href="https://2014.igem.org/Team:Goettingen/notebook_wetlab" class="button_pre"><b>Previous</b></a><a href="https://2014.igem.org/Team:Goettingen/notebook_wetlab/gfp" class="button_next"><b>Next</b></a></div>
                 </div>
                 </div>

Latest revision as of 23:50, 17 October 2014

Notebook


July


- Used BioBricks from library:
- Tick with a tip a hole in plate and resolve the pellet in 10µl sterile water, red color was visible, pipeting up and down, wait 5 minutes and put 10 µl into a PCR cup and freeze it
- 1 µl is transformed into DH5α → plated on Cm or kan plates
- Transformation worked, pick colonies for o/n in LB + Cm to isolate BB-plasmids
- Isolation of plasmids

Agarose gel (1%):

August


→After several tries to construct our BioBricks on the described way we changed to seamless cloning technique

- PCRs for seamless cloning:

Agarose gels (1%):
- Purification of the products and measurement of the concentration of each product.
- Amplification of Leu2, Trp1 and 2-micron with 2 sets of primers to create mutated genes that loose restriction sites:

1. PCR to create for each gene 2 fragments that contain 15 bp overhangs to the other one.
2. Use fusion PCR approach to fuse genes with silent mution together.
3. Gel extract the rightly fused PCR product and use it in a normal PCR for amplification.
- Purification and sequencing of this three samples


September


- results sequencing:

Trp1 AGGCCGCAGAATGTGCTCTGGATTCCGATGCTGACTTGCT
Trp1*AGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCT

Leu2 TGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAA
Leu2*TGGTCCTAAATGGGGTACAGGTAGTGTTAGACCTGAACAA

2-micron GAAGTTCCTATACTTTCTAG****AGAATAGGAACTTCGGAATAGGAACTTC
2-micron*GAAGTTCCTATACTTTCTAGCTAGAGAATAGGAACTTCGGAATAGGAACTTC

⇒ silent mutations delete the restriction sides

- Prepare seamless mixture (see protocol and formula) with I – VI
- Transformation of the whole reaction sample into DH5α.
- Grown colonies on plates → pick several colonies and grow over night.
- Colony PCR with primer on backbone and primer in insert
- plasmid isolation of positives

- Amplification of construct via PCR
- Gel extraction & Purification & seamless cloning with A-F
- Transformation into DH5α
- Pick colonies and isolate plasmids from overnight culture
- colony PCR to verify construct
- restriction of the two different plasmids and the fusion product of 2-micron ligation over night of the 2-micron behind restriction site in plasmid backbones
- Transformation into DH5α.
- Pick several colonies into 4 mL LB +Cm for overnight cultures.
- Colony PCR and isolation of plasmids.
- Chose positives for further work and shipping to the registry

Retrieved from "http://2014.igem.org/Team:Goettingen/notebook_wetlab/biobricks"