Team:Harvard/color/notebook

From 2010.igem.org

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         <tr><td class="notebook">Week 7</td><td class="notebook"><a class"notebooklink" href="#07-26-2010">07-26-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-27-2010" class="notebooklink">07-27-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-28-2010">07-28-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-29-2010">07-29-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-30-2010">07-30-2010</a></td></tr>
         <tr><td class="notebook">Week 7</td><td class="notebook"><a class"notebooklink" href="#07-26-2010">07-26-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-27-2010" class="notebooklink">07-27-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-28-2010">07-28-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-29-2010">07-29-2010</a></td><td class="notebook"><a class="notebooklink" href="#07-30-2010">07-30-2010</a></td></tr>
 +
 +
        <tr><td class="notebook">Week 8</td><td class="notebook">-</td><td class="notebook">-</td><td class="notebook"><a class="notebooklink" href="#08-04-2010">08-04-2010</a></td><td class="notebook"><a class="notebooklink" href="#08-05-2010">08-05-2010</a></td><td class="notebook"><a class="notebooklink" href="#08-06-2010">08-06-2010</a></td></tr>
</table>
</table>
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<li>Ran 4 ul of PCR reaction on a 1% agarose gel</li>
<li>Ran 4 ul of PCR reaction on a 1% agarose gel</li>
<div><a href="https://static.igem.org/mediawiki/2010/0/0b/072610amipcr1_ann.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/0/0b/072610amipcr1_ann.png"  width="100px"/> [click to enlarge]</a></div>
<div><a href="https://static.igem.org/mediawiki/2010/0/0b/072610amipcr1_ann.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/0/0b/072610amipcr1_ann.png"  width="100px"/> [click to enlarge]</a></div>
-
</ul>
 
<li>Bands appeared to be the correct size, so started PCR stitching process</li>
<li>Bands appeared to be the correct size, so started PCR stitching process</li>
<li>Ran two PCR reactions, one for each of LUT2 and BETA-OHASE 1</li>
<li>Ran two PCR reactions, one for each of LUT2 and BETA-OHASE 1</li>
<li class="indent">Template: Rxn 1, 2, and 3 of the appropriate construct</li>
<li class="indent">Template: Rxn 1, 2, and 3 of the appropriate construct</li>
 +
<li class="indent">Template diluted to 1 ng/ul, used 1 ul of each template</li>
<li class="indent">Primers: A and B for both reactions</li>
<li class="indent">Primers: A and B for both reactions</li>
 +
<li class="indent">Size expected to be around 450 bp</li>
<li>PCR purified and ran 4 ul on a 1% agarose gel</li>
<li>PCR purified and ran 4 ul on a 1% agarose gel</li>
<div><a href="https://static.igem.org/mediawiki/2010/c/c9/072610pcr2.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/c/c9/072610pcr2.png" width="100px"/> [click to enlarge]</a></div>
<div><a href="https://static.igem.org/mediawiki/2010/c/c9/072610pcr2.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/c/c9/072610pcr2.png" width="100px"/> [click to enlarge]</a></div>
 +
<li>PCR was not clean, decided to re-do stitching reaction with undiluted template (concentrations around 50 ng/ul, used 1 ul of each</li>
 +
<li>Ran 4 ul on 1% agarose gel</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/8/8b/072610amirnapcr3_ann.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/8/8b/072610amirnapcr3_ann.png"  width="100px"/> [click to enlarge]</a></div>
 +
<li>Observed clear band at correct location for BETA-OHASE 1, but not for LUT2</li>
 +
</ul>
</div>
</div>
 +
<div id="entry">
 +
<span id="date"><a name="07-27-2010" class="date">07-27-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/>
 +
<ul>
 +
<li>Redid PCR stitching for LUT2, ran 4 ul on a gel</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/1/1e/072710lut2pcrstitch.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/1/1e/072710lut2pcrstitch.png"  width="100px"/> [click to enlarge]</a></div>
 +
<li>Observed clear bands, including one in the right location</li>
 +
<li>Ran entire reaction on a gel (half in each lane), cut out appropriate band and gel purified</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/8/84/072710lut2pcrstitchgelpurification-1.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/8/84/072710lut2pcrstitchgelpurification-1.png"  width="100px"/> [click to enlarge]</a></div>
 +
<li>Digested gel purified LUT2, PCR purified BETA-OHASE 1, and B21 (Biobrick part in V0120 - to serve as backbone) with EcoRI and PstI to prepare for ligation of constructs into biobrick backbone</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/3/36/07272010pcrdigest_ann.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/3/36/07272010pcrdigest_ann.png"  width="100px"/> [click to enlarge]</a></div>
 +
<li>LUT2 had the correctly sized band, but BETA-OHASE 1 had two bands, one at 400 and one at 100.</li>
 +
<li>Checked sequences of primers, and determined that primers for BETA-OHASE 1 contained an EcoRI site</li>
 +
<li>Ordered new primers without biobrick restriction sites for BETA-OHASE 1. Checked other primers for restriction sites, and found them to be clean.</li>
 +
</ul>
 +
</div>
 +
 +
<div id="entry">
 +
<span id="date"><a name="07-28-2010" class="date">07-28-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/>
 +
<ul>
 +
<li>Gel purified LUT2 and top band from B21 (backbone)</li>
 +
<li>Ligated LUT2 and backbone. Transformed into TURBO e. coli and plated on LB + Amp.</li>
 +
</ul>
 +
</div>
 +
 +
<div id="entry">
 +
<span id="date"><a name="07-29-2010" class="date">07-29-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/>
 +
<ul>
 +
<li>Obtained colonies for LUT2. Set up four cultures in LB + Amp.</li>
 +
</ul>
 +
</div>
 +
 +
<div id="entry">
 +
<span id="date"><a name="08-04-2010" class="date">08-04-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/>
 +
<ul>
 +
<li>Received new primers for BETA-OHASE 1 - began building knockdown constructs for that and LYC</li>
 +
<li>PCR reaction on RS300 to insert recognition sites. Primers listed below</li>
 +
<li class="indent">LYC rxn 1: A, CP12</li>
 +
<li class="indent">LYC rxn 2: CP10, CP11</li>
 +
<li class="indent">LYC rxn 3: CP9, B</li>
 +
<li class="indent">BETA-OHASE 1 rxn 1: A, CP16</li>
 +
<li class="indent">BETA-OHASE 1 rxn 2: CP14, CP15</li>
 +
<li class="indent">BETA-OHASE 1 rxn 3: CP13, B</li>
 +
</ul>
 +
</div>
 +
 +
 +
<div id="entry">
 +
<span id="date"><a name="08-05-2010" class="date">08-05-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/>
 +
<b>PCR Stitching of LYC and BETA-OHASE 1 amiRNA parts; Digestion and Ligation into biobrick backbone</b>
 +
<ul>
 +
<li>Ran PCR reaction on a 1% agarose gel.</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/1/15/080510lycbohpcrparts_ann.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/1/15/080510lycbohpcrparts_ann.png"  width="100px"/> [click to enlarge]</a></div>
 +
<li>Initial PCR was successful. Gel purified parts</li>
 +
<li>Performed PCR Stitching for each of BETA-OHASE 1 and LYC</li>
 +
<li class="indent">Template: Rxn 1, 2, and 3 of the appropriate construct</li>
 +
<li class="indent">Used 1 ul of each template - concentrations were all around 25 ng/ul</li>
 +
<li class="indent">Primers: A and B for both reactions</li>
 +
<li class="indent">Size expected to be around 450 bp</li>
 +
<li>PCR purified and ran 4 ul on a 1% agarose gel</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/8/80/080510lycbohpcrstitch_ann.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/8/80/080510lycbohpcrstitch_ann.png"  width="100px"/> [click to enlarge]</a></div>
 +
<li>PCR was successful. Gel purified products</li>
 +
<li>Digested products with EcoRI and PstI to ligate into V0120 backbone.</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/8/8d/08052010lycbetadigest.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/8/8d/08052010lycbetadigest.png"  width="100px"/> [click to enlarge]</a></div>
 +
<li>Successful digestion - gel purified and ligated into backbone digested EcoRI/PstI last week</li>
 +
<li>Transformed into TURBO E. coli and plated on LB + Amp</li>
 +
</ul>
 +
<b>PCR Amplification of petal promoters</b>
 +
<ul>
 +
<li>Ran PCR to amplify petal promoters</li>
 +
<li class="indent">Template: Arabidopsis genomic DNA, 80ng</li>
 +
<li class="indent">Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)</li>
 +
<li class="indent">Protocol: Phusion</li>
 +
<li>Ran completed reaction on 1% agarose gel</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/a/a8/080510failedpromoterpcr.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/a/a8/080510failedpromoterpcr.png" width="100px" /> [click to enlarge]</a></div>
 +
<li>PCR Failed. Retry with PFx</li>
 +
<li class="indent">Template: Arabidopsis genomic DNA, 80ng</li>
 +
<li class="indent">Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)</li>
 +
<li class="indent">Protocol: PFx, extension time 2 min</li>
 +
<li>Ran completed reaction on 1% agarose gel</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/3/32/08052010promoterpcr.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/3/32/08052010promoterpcr.png"  width="100px"/> [click to enlarge]</a></div>
 +
</ul>
 +
</div>
 +
 +
<div id="entry">
 +
<span id="date"><a name="08-06-2010" class="date">08-06-2010</a></span> [<a class="top" href="#notebook">top</a>]<hr/>
 +
<ul>
 +
<li>Got colonies for C3 (LYC) but not for C2 (BETA-OHASE 1). Picked four colonies and set up cultures for C3</li>
 +
<li>Redid digestion on C2, and digested PCR products C4-6 with EcoRI and PstI. Also digested B21 with EcoRI and PstI with TSAP to obtain backbone.</li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/4/45/080610pcrdigest1.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/4/45/080610pcrdigest1.png" width="100px" /> [click to enlarge]</a></div>
 +
<li>C2 and B21 digests worked, C4 had a PstI site inside the part and C5/C6 had EcoRI sites</li>
 +
<li>Gel purified C2 and B21 (top) bands</li>
 +
<li>Redigested C4-C6 with XbaI and SpeI, as well as B21 with XbaI and SpeI with TSAP to obtain backbone<li>
 +
<div><a href="https://static.igem.org/mediawiki/2010/a/a0/080610pcrdigest2.png" id="single_image"><img src="https://static.igem.org/mediawiki/2010/a/a0/080610pcrdigest2.png" width="100px" /> [click to enlarge]</a></div>
 +
<li>Gel ran strangely - ladder didn't show. Cut out bands and gel purified, then ran second gel to check sizes again</li>
 +
 +
<li>Miniprepped cultures set up in the morning, and sent out for sequencing</li>
 +
</ul>
 +
</div>
</html>
</html>

Latest revision as of 03:43, 23 October 2010



notebook

Week 6----07-23-2010
Week 707-26-201007-27-201007-28-201007-29-201007-30-2010
Week 8--08-04-201008-05-201008-06-2010


07-23-2010 [top]
  • Received primers for LUT2 and BETA-OHASE 1 - began building knockdown constructs
  • PCR reaction on RS300 to insert recognition sites. Primers listed below
  • LUT2 rxn 1: A, CP4
  • LUT2 rxn 2: CP2, CP3
  • LUT2 rxn 3: CP1, B
  • BETA-OHASE 1 rxn 1: A, CP8
  • BETA-OHASE 1 rxn 2: CP6, CP7
  • BETA-OHASE 1 rxn 3: CP5, B
  • PCR purified completed reaction
07-26-2010 [top]
  • Ran 4 ul of PCR reaction on a 1% agarose gel
  • Bands appeared to be the correct size, so started PCR stitching process
  • Ran two PCR reactions, one for each of LUT2 and BETA-OHASE 1
  • Template: Rxn 1, 2, and 3 of the appropriate construct
  • Template diluted to 1 ng/ul, used 1 ul of each template
  • Primers: A and B for both reactions
  • Size expected to be around 450 bp
  • PCR purified and ran 4 ul on a 1% agarose gel
  • PCR was not clean, decided to re-do stitching reaction with undiluted template (concentrations around 50 ng/ul, used 1 ul of each
  • Ran 4 ul on 1% agarose gel
  • Observed clear band at correct location for BETA-OHASE 1, but not for LUT2
07-27-2010 [top]
  • Redid PCR stitching for LUT2, ran 4 ul on a gel
  • Observed clear bands, including one in the right location
  • Ran entire reaction on a gel (half in each lane), cut out appropriate band and gel purified
  • Digested gel purified LUT2, PCR purified BETA-OHASE 1, and B21 (Biobrick part in V0120 - to serve as backbone) with EcoRI and PstI to prepare for ligation of constructs into biobrick backbone
  • LUT2 had the correctly sized band, but BETA-OHASE 1 had two bands, one at 400 and one at 100.
  • Checked sequences of primers, and determined that primers for BETA-OHASE 1 contained an EcoRI site
  • Ordered new primers without biobrick restriction sites for BETA-OHASE 1. Checked other primers for restriction sites, and found them to be clean.
07-28-2010 [top]
  • Gel purified LUT2 and top band from B21 (backbone)
  • Ligated LUT2 and backbone. Transformed into TURBO e. coli and plated on LB + Amp.
07-29-2010 [top]
  • Obtained colonies for LUT2. Set up four cultures in LB + Amp.
08-04-2010 [top]
  • Received new primers for BETA-OHASE 1 - began building knockdown constructs for that and LYC
  • PCR reaction on RS300 to insert recognition sites. Primers listed below
  • LYC rxn 1: A, CP12
  • LYC rxn 2: CP10, CP11
  • LYC rxn 3: CP9, B
  • BETA-OHASE 1 rxn 1: A, CP16
  • BETA-OHASE 1 rxn 2: CP14, CP15
  • BETA-OHASE 1 rxn 3: CP13, B
08-05-2010 [top]
PCR Stitching of LYC and BETA-OHASE 1 amiRNA parts; Digestion and Ligation into biobrick backbone
  • Ran PCR reaction on a 1% agarose gel.
  • Initial PCR was successful. Gel purified parts
  • Performed PCR Stitching for each of BETA-OHASE 1 and LYC
  • Template: Rxn 1, 2, and 3 of the appropriate construct
  • Used 1 ul of each template - concentrations were all around 25 ng/ul
  • Primers: A and B for both reactions
  • Size expected to be around 450 bp
  • PCR purified and ran 4 ul on a 1% agarose gel
  • PCR was successful. Gel purified products
  • Digested products with EcoRI and PstI to ligate into V0120 backbone.
  • Successful digestion - gel purified and ligated into backbone digested EcoRI/PstI last week
  • Transformed into TURBO E. coli and plated on LB + Amp
PCR Amplification of petal promoters
  • Ran PCR to amplify petal promoters
  • Template: Arabidopsis genomic DNA, 80ng
  • Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)
  • Protocol: Phusion
  • Ran completed reaction on 1% agarose gel
  • PCR Failed. Retry with PFx
  • Template: Arabidopsis genomic DNA, 80ng
  • Primers: CP17/CP18 (At1G11850), CP19/CP20 (At1G70720 1.1kb), CP21/CP22 (At1G70720 1.5kb)
  • Protocol: PFx, extension time 2 min
  • Ran completed reaction on 1% agarose gel
08-06-2010 [top]
  • Got colonies for C3 (LYC) but not for C2 (BETA-OHASE 1). Picked four colonies and set up cultures for C3
  • Redid digestion on C2, and digested PCR products C4-6 with EcoRI and PstI. Also digested B21 with EcoRI and PstI with TSAP to obtain backbone.
  • C2 and B21 digests worked, C4 had a PstI site inside the part and C5/C6 had EcoRI sites
  • Gel purified C2 and B21 (top) bands
  • Redigested C4-C6 with XbaI and SpeI, as well as B21 with XbaI and SpeI with TSAP to obtain backbone
  • Gel ran strangely - ladder didn't show. Cut out bands and gel purified, then ran second gel to check sizes again
  • Miniprepped cultures set up in the morning, and sent out for sequencing

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