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Team:TU Darmstadt/Notebook/Labjournal/K1497001
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| - | <!--TYPO3SEARCH_begin--><div id="c111" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">K1497001 - Chalcone Synthase (CHS)</h1></div><div class="csc-textpic-text"><p>We kindly received the CHS gene from Dr. Stefan Martens. We introduced Biobrick prefix and suffix and amplified our part of interest via plasmid PCR using the primers "CHS_psb1c3_Pre2" and "CHS_pscb1c3_Suf2" Subsequently we cleaned up the PCR product by DpnI digestion and Wizard PCR Clean Up Kit. Afterwards we cut the PCR product with EcoRI + PstI and cloned the insert into a previously digestion of mRFP-pSB1C3 with EcoRI + PstI. We used T4 ligase for ligation and transformed the ligation into Top10 E.coli via heat shock. For spreading out we used CMP-LB agar plates. After incubating overnight at 37°C we analyzed obtained colonies by colony-PCR and agarose gel electrophoresis. We inoculated positive colonies in 5 ml LB with CMP. After incubating for 16 h at 37°C we isolated the plasmids. For further verification we send the samples to Eurofins for sequencing.</p></div></div><!--TYPO3SEARCH_end--> | + | <!--TYPO3SEARCH_begin--><div id="c111" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">K1497001 - Chalcone Synthase (CHS)</h1></div><div class="csc-textpic-text"><p>We kindly received the CHS gene from Dr. Stefan Martens. We introduced Biobrick prefix and suffix and amplified our part of interest via plasmid PCR using the primers "CHS_psb1c3_Pre2" and "CHS_pscb1c3_Suf2" Subsequently we cleaned up the PCR product by DpnI digestion and Wizard PCR Clean Up Kit. Afterwards we cut the PCR product with EcoRI + PstI and cloned the insert into a previously digestion of mRFP-pSB1C3 with EcoRI + PstI. We used T4 ligase for ligation and transformed the ligation into Top10 E.coli via heat shock. For spreading out we used CMP-LB agar plates. After incubating overnight at 37°C we analyzed obtained colonies by colony-PCR and agarose gel electrophoresis. We inoculated positive colonies in 5 ml LB with CMP. After incubating for 16 h at 37°C we isolated the plasmids gaining the BioBrick <a href="http://parts.igem.org/Part:BBa_K1497001" title="Opens internal link in current window" class="internal-link"> BBa_K1497001</a>. <br> For further verification we send the samples to Eurofins for sequencing.</p></div></div><!--TYPO3SEARCH_end--> |
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Latest revision as of 18:46, 17 October 2014
K1497001 - Chalcone Synthase (CHS)
We kindly received the CHS gene from Dr. Stefan Martens. We introduced Biobrick prefix and suffix and amplified our part of interest via plasmid PCR using the primers "CHS_psb1c3_Pre2" and "CHS_pscb1c3_Suf2" Subsequently we cleaned up the PCR product by DpnI digestion and Wizard PCR Clean Up Kit. Afterwards we cut the PCR product with EcoRI + PstI and cloned the insert into a previously digestion of mRFP-pSB1C3 with EcoRI + PstI. We used T4 ligase for ligation and transformed the ligation into Top10 E.coli via heat shock. For spreading out we used CMP-LB agar plates. After incubating overnight at 37°C we analyzed obtained colonies by colony-PCR and agarose gel electrophoresis. We inoculated positive colonies in 5 ml LB with CMP. After incubating for 16 h at 37°C we isolated the plasmids gaining the BioBrick BBa_K1497001.
For further verification we send the samples to Eurofins for sequencing.
