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Team:TU Darmstadt/Notebook/Labjournal/K1497024
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| - | <!--TYPO3SEARCH_begin--><div id="c326" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Scaffold GBD domain</h1></div><p>The PCR -amplification of the single domains of the <i>scaffold</i> protein yielded strong and clear bands migrating at the expected heights (see figuree 1). SH3 has a full length of 286 bp, PDZ of 400 bp and GBD of 354 bp. The digested fragments were cloned into the vector pSB1C3. Successful insertion of the SH3 and PDZ domain was verified by a colony PCR ( see figure 2). All three tested colonies were positive. However, all bands of the GBD domain differed from each other. Only one of three tested PCR -reactions showed a weak intensity at the expected height. Verification through sequencing of the generated vectors was omitted, since the three domains were initially amplified from positive sequenced template plasmids. The PCR-products were cloned into the vector pSB1C3 and E. coli Top10 cells were transformed with the constructs and tested via colongy-PCR. </p></div><div id="c327" class="csc-default"><div class="csc-textpic csc-textpic-center csc-textpic-above"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><div class="csc-textpic-center-outer"><div class="csc-textpic-center-inner"><figure class="csc-textpic-image csc-textpic-last"><img src=" | + | <!--TYPO3SEARCH_begin--><div id="c326" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Scaffold GBD domain</h1></div><p>The PCR -amplification of the single domains of the <i>scaffold</i> protein yielded strong and clear bands migrating at the expected heights (see figuree 1). SH3 has a full length of 286 bp, PDZ of 400 bp and GBD of 354 bp. The digested fragments were cloned into the vector pSB1C3. Successful insertion of the SH3 and PDZ domain was verified by a colony PCR ( see figure 2). All three tested colonies were positive. However, all bands of the GBD domain differed from each other. Only one of three tested PCR -reactions showed a weak intensity at the expected height. Verification through sequencing of the generated vectors was omitted, since the three domains were initially amplified from positive sequenced template plasmids. The PCR-products were cloned into the vector pSB1C3 and E. coli Top10 cells were transformed with the constructs and tested via colongy-PCR. </p></div><div id="c327" class="csc-default"><div class="csc-textpic csc-textpic-center csc-textpic-above"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><div class="csc-textpic-center-outer"><div class="csc-textpic-center-inner"><figure class="csc-textpic-image csc-textpic-last"><img src="https://static.igem.org/mediawiki/parts/5/5f/Scaffold_single_domains.png" width="337" height="229" alt=""></figure></div></div></div><div class="csc-textpic-text"><p>Figure 1: Standard PCR of the single subdomains of the scaffold protein. SH3 (left) has a uncutted size of 286 bp, PDZ (middle) of 400 bp and GBD (right) of 354 bp. The 2-log ladder (M) was applied on the left side.</p></div></div></div><div id="c329" class="csc-default"><div class="csc-textpic csc-textpic-center csc-textpic-above"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><div class="csc-textpic-center-outer"><div class="csc-textpic-center-inner"><figure class="csc-textpic-image csc-textpic-last"><img src="https://static.igem.org/mediawiki/parts/7/7e/Scaffold_single_domains_2.png" width="304" height="265" alt=""></figure></div></div></div><div class="csc-textpic-text"><p style="text-align:justify"><span lang="EN-IE">Figure 2: Colony PCR of the single domains cloned into the vector pSB1C3. In each instance three colonies were analyzed. SH3 has a undigested size of 286 bp, PDZ (middle) of 400 bp and GBD (right) of 354 bp. The 2-log ladder (M) was applied on the left side.</span></p></div></div></div><!--TYPO3SEARCH_end--> |
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Revision as of 23:58, 15 October 2014
Scaffold GBD domain
The PCR -amplification of the single domains of the scaffold protein yielded strong and clear bands migrating at the expected heights (see figuree 1). SH3 has a full length of 286 bp, PDZ of 400 bp and GBD of 354 bp. The digested fragments were cloned into the vector pSB1C3. Successful insertion of the SH3 and PDZ domain was verified by a colony PCR ( see figure 2). All three tested colonies were positive. However, all bands of the GBD domain differed from each other. Only one of three tested PCR -reactions showed a weak intensity at the expected height. Verification through sequencing of the generated vectors was omitted, since the three domains were initially amplified from positive sequenced template plasmids. The PCR-products were cloned into the vector pSB1C3 and E. coli Top10 cells were transformed with the constructs and tested via colongy-PCR.
Figure 1: Standard PCR of the single subdomains of the scaffold protein. SH3 (left) has a uncutted size of 286 bp, PDZ (middle) of 400 bp and GBD (right) of 354 bp. The 2-log ladder (M) was applied on the left side.
Figure 2: Colony PCR of the single domains cloned into the vector pSB1C3. In each instance three colonies were analyzed. SH3 has a undigested size of 286 bp, PDZ (middle) of 400 bp and GBD (right) of 354 bp. The 2-log ladder (M) was applied on the left side.
