Team:TU Darmstadt/Notebook/Labjournal/K1497027

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<p>Sie sind hier:&nbsp; <a href="https://2014.igem.org/Team:TU_Darmstadt" >wiki</a> &rsaquo;&nbsp;<a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook" >Notebook</a> &rsaquo;&nbsp;<span class="current"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Labjournal" >Labjournal</a>&rsaquo;&nbsp;<span class="current"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Labjournal/K1497027" >K1497027</a></span></p>
 
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<p>&nbsp;</p></div><div id="c377" class="csc-default"><div class="csc-textpic csc-textpic-intext-right"><div class="csc-textpic-imagewrap" data-csc-images="1" data-csc-cols="2"><figure class="csc-textpic-image csc-textpic-last"><img src="https://static.igem.org/mediawiki/parts/f/fb/Doppeldomaenen2.PNG" width="300" height="279" alt=""></figure></div><div class="csc-textpic-text"><p>Figure 1: Backbone production for scaffold double domain vectors.
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<p>The plasmids pSB1C3-GBD , pSB1C3-PDZ, and pSB1C3-SH3 were digested with <i>BamHI</i> and <i>PstI</i>. Only names of the respective domains are shown. A sample of the undigested vector (left) was loaded next to the digested vectors (right). The digested vectors migrate at their expected heights, as well as the initial vectors. The 2-log marker (M) is shown on the left.</p></div></div></div><div id="c378" 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/f/f4/Doppeldomaenen_1.PNG" width="600" height="248" alt=""></figure></div></div></div><div class="csc-textpic-text"><p>Figure 2: Colony PCRs after transformation of <i>E. coli</i> Top10 cells with the ligation relations.
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<p>Figure 1: Backbone production for scaffold double domain vectors.</p>
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<p>The plasmids pSB1C3-GBD , pSB1C3-PDZ, and pSB1C3-SH3 were digested with <i>BamHI</i> and <i>PstI</i>. Only names of the respective domains are shown. A sample of the undigested vector (left) was loaded next to the digested vectors (right). The digested vectors migrate at their expected heights, as well as the initial vectors. The 2-log marker (M) is shown on the left.</p><
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<p>Figure 2: Colony PCRs after transformation of <i>E. coli</i> Top10 cells with the ligation relations.
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<p>Triplicates of the PCR reactions for the obtained double domains are shown. The 2-log marker (M) is shown on the left. Kolonie 2 (middle) was always positive for all constructs.</p></div></div></div><!--TYPO3SEARCH_end-->
<p>Triplicates of the PCR reactions for the obtained double domains are shown. The 2-log marker (M) is shown on the left. Kolonie 2 (middle) was always positive for all constructs.</p></div></div></div><!--TYPO3SEARCH_end-->
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Latest revision as of 15:50, 17 October 2014

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Construction of scaffold double domain vectors in pSB1C3

Two single domains of the scaffold protein were joined together by inserting a single domain into the earlier constructed single domain vectors. The backbone vectors were digested with BamHI and PstI. The inserts were amplified with primer containing the BglII, EcoRI, and XbaI restriction site and the BamHI, SpeI, and PstI hestrictions site. The backbones were ligated with the inserts and Top10 Zells were transformed with the products. The colonies were screent via colonie PCR and positive colones were grown in LB-Cmp-Medium. The plasmids were recoverd via Mini-Plasmid-Preparation.

 

 

Figure 1: Backbone production for scaffold double domain vectors.

The plasmids pSB1C3-GBD , pSB1C3-PDZ, and pSB1C3-SH3 were digested with BamHI and PstI. Only names of the respective domains are shown. A sample of the undigested vector (left) was loaded next to the digested vectors (right). The digested vectors migrate at their expected heights, as well as the initial vectors. The 2-log marker (M) is shown on the left.

<

Figure 2: Colony PCRs after transformation of E. coli Top10 cells with the ligation relations.

Triplicates of the PCR reactions for the obtained double domains are shown. The 2-log marker (M) is shown on the left. Kolonie 2 (middle) was always positive for all constructs.