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Team:TU Darmstadt/Notebook/Labjournal/K1497000
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| - | <!--TYPO3SEARCH_begin--><div id="c110" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader | + | <!--TYPO3SEARCH_begin--><div id="c110" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">K1497000 - Chalcone Isomerase (CHI)</h1></div><div class="csc-textpic-text"><div><p>We kindly received the CHI on a TOPO-backbone from Dr. Stefan Martens. We isolated the gene with plasmid PCR using primer containing BioBrick prefix (CHI_psb1c3_Pre) and suffix (CHI_psb1c3_Suf2). We digested the PCR product with DpnI and incubated for 30 min. Afterwards we purified the sample. We verified the desired DNA length via an agarose gel. We cut the clean sample once with EcoRI and PstI and cloned the digestion into a pSB1A2 backbone. We used T4 ligase for ligation and Top10 E.coli cells for transformation. Finally we spread out on AMP LB agar. The CHI coding part contained two restriction sites (2x XbaI), that are not compatible with the Biobrick-system. So we had to edit the gene via QuickChange. For this reason we used <a href="http://sevierlab.vet.cornell.edu/resources/Stratagene-QuikchangeManual.pdf" target="_blank">QuikChange® Site-Directed Mutagenesis Kit from Stratagene</a> with "QCI_CHI_F" and "QCI_CHI_R" primers. We analyzed the PCR products via agarose gel electrophoresis (Figure 1) and cleaned positive samples with DpnI digestion and Wizard PCR Clean Up Kit (Promega). Afterwards we transformed the plasmid into Top10 cells via heatshock and spread them on LB plates with AMP. After incubating the plates overnight, we inoculated 2 colonies in 5 ml LB with AMP. After 16 hours at 37°C, we purified plasmids of two colonies. We eluted the plasmid DNA in 50 µl nuclease free H2O and obtained a plasmid concentration of 26,4 ng/µL. We verified the success of the QuickChange PCR by cutting the obtained plasmids with XbaI and expecting 2 fragments. We repeated the QuickChange step for the second XbaI restriction site with "QCIII_CHI_F" and "QCIII_CHI_R" primers and cut the obtained plasmid with EcoRI + PstI. We ligated the restriction's insert with a pSB1C3 backbone by using T4 Ligase. Afterwards we transformated the ligation in Top10 E.coli. We gained the backbone from mRFP-pSB1C3 by cutting with EcoRI and PstI. Using a mRFP-insert allowed us to select colonies in LB agar plates easily by considering only white colonies for colony-PCR. We analyzed obtanied white colonies by colony-PCR and agarose gel electrophoresis (Figure 2). We inoculated each positive colony in 5 ml LB and purified plasmids after incubating for 16h at 37°C gaining the BioBrick <a href="http://parts.igem.org/Part:BBa_K1497000" title="Opens internal link in current window" class="internal-link"> BBa_K1497000</a>. </p></div></div></div> |
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Revision as of 18:45, 17 October 2014
K1497000 - Chalcone Isomerase (CHI)
We kindly received the CHI on a TOPO-backbone from Dr. Stefan Martens. We isolated the gene with plasmid PCR using primer containing BioBrick prefix (CHI_psb1c3_Pre) and suffix (CHI_psb1c3_Suf2). We digested the PCR product with DpnI and incubated for 30 min. Afterwards we purified the sample. We verified the desired DNA length via an agarose gel. We cut the clean sample once with EcoRI and PstI and cloned the digestion into a pSB1A2 backbone. We used T4 ligase for ligation and Top10 E.coli cells for transformation. Finally we spread out on AMP LB agar. The CHI coding part contained two restriction sites (2x XbaI), that are not compatible with the Biobrick-system. So we had to edit the gene via QuickChange. For this reason we used QuikChange® Site-Directed Mutagenesis Kit from Stratagene with "QCI_CHI_F" and "QCI_CHI_R" primers. We analyzed the PCR products via agarose gel electrophoresis (Figure 1) and cleaned positive samples with DpnI digestion and Wizard PCR Clean Up Kit (Promega). Afterwards we transformed the plasmid into Top10 cells via heatshock and spread them on LB plates with AMP. After incubating the plates overnight, we inoculated 2 colonies in 5 ml LB with AMP. After 16 hours at 37°C, we purified plasmids of two colonies. We eluted the plasmid DNA in 50 µl nuclease free H2O and obtained a plasmid concentration of 26,4 ng/µL. We verified the success of the QuickChange PCR by cutting the obtained plasmids with XbaI and expecting 2 fragments. We repeated the QuickChange step for the second XbaI restriction site with "QCIII_CHI_F" and "QCIII_CHI_R" primers and cut the obtained plasmid with EcoRI + PstI. We ligated the restriction's insert with a pSB1C3 backbone by using T4 Ligase. Afterwards we transformated the ligation in Top10 E.coli. We gained the backbone from mRFP-pSB1C3 by cutting with EcoRI and PstI. Using a mRFP-insert allowed us to select colonies in LB agar plates easily by considering only white colonies for colony-PCR. We analyzed obtanied white colonies by colony-PCR and agarose gel electrophoresis (Figure 2). We inoculated each positive colony in 5 ml LB and purified plasmids after incubating for 16h at 37°C gaining the BioBrick BBa_K1497000.
Figure 1: Quickchange of CHI. On the right we applied GeneRuler DNA Ladder Mix. The product should have a length around 3,0 kbp.
Figure 2: Colony PCR of CHI with VF2- and VR- primers. As ladder we addes GeneRuler DNA ladder mix. The product should have a length of 1,1 kbp.
