Team:Goettingen/NoteBook w7
From 2013.igem.org
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- | <div class=" | + | |
+ | |||
+ | <div class="tlob" id="tl_0718"> | ||
+ | <span class="date">18th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title goe-rt"> Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10), Colony PCR for DarR and Riboswitch biobrick transformation from 15.7.13</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <div class="Section1" style="layout-grid:15.6pt"> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Gel | ||
+ | run: Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to | ||
+ | RT-C10)<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Addition of 5 µl 5xLD to the | ||
+ | samples</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Loading of 5 µl of each sample | ||
+ | on 1 % agarose gel</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Loading of 3 µl 2 log ladder as | ||
+ | a marker</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Gel run at 100 V</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">EtBr staining + destaining in | ||
+ | water</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">UV detection</span></p> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="mso-no-proof:yes"><!--[if gte vml 1]><v:shapetype id="_x0000_t75" | ||
+ | coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe" | ||
+ | filled="f" stroked="f"> | ||
+ | <v:stroke joinstyle="miter"/> | ||
+ | <v:formulas> | ||
+ | <v:f eqn="if lineDrawn pixelLineWidth 0"/> | ||
+ | <v:f eqn="sum @0 1 0"/> | ||
+ | <v:f eqn="sum 0 0 @1"/> | ||
+ | <v:f eqn="prod @2 1 2"/> | ||
+ | <v:f eqn="prod @3 21600 pixelWidth"/> | ||
+ | <v:f eqn="prod @3 21600 pixelHeight"/> | ||
+ | <v:f eqn="sum @0 0 1"/> | ||
+ | <v:f eqn="prod @6 1 2"/> | ||
+ | <v:f eqn="prod @7 21600 pixelWidth"/> | ||
+ | <v:f eqn="sum @8 21600 0"/> | ||
+ | <v:f eqn="prod @7 21600 pixelHeight"/> | ||
+ | <v:f eqn="sum @10 21600 0"/> | ||
+ | </v:formulas> | ||
+ | <v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/> | ||
+ | <o:lock v:ext="edit" aspectratio="t"/> | ||
+ | </v:shapetype><v:shape id="图片_x0020_1" o:spid="_x0000_i1027" type="#_x0000_t75" | ||
+ | style='width:471pt;height:481.5pt;visibility:visible;mso-wrap-style:square'> | ||
+ | <v:imagedata src="raw-07.18.files/image001.png" o:title="" croptop="-1952f" | ||
+ | cropbottom="-85f" cropleft="-1113f" cropright="-1374f"/> | ||
+ | </v:shape><![endif]--><!--[if !vml]--><img width="500" src="https://static.igem.org/mediawiki/2013/thumb/8/88/Goe-18.07.13-RT-1.png/586px-Goe-18.07.13-RT-1.png" v:shapes="图片_x0020_1"><!--[endif]--></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal"><span lang="EN-US">Re-Trafo Clones 1, 4 – 7, 9 and 10 look | ||
+ | promising: could contain RBS-GFP-Terminator construct in pSB1A3 backbone</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings; | ||
+ | mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><span lang="EN-US">Re-Trafo Clone 2 has a strange additional band (from plasmid?)</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings; | ||
+ | mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><span lang="EN-US">Re-Trafo Clone 8: no product</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings; | ||
+ | mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Even for Plasmid of | ||
+ | RBS-GFP-Terminator C12, the additional band at the height of the terminator PCR | ||
+ | product is not seen </span></b><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-font-family: | ||
+ | Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> but for plasmid GFP this band could be visible… additionally strong | ||
+ | band of GFP-Terminator C1 PCR product runs higher than band of PCR product for | ||
+ | RBS-GFP-Terminator C12 </span></b><b style="mso-bidi-font-weight:normal"><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family:"Times New Roman"; | ||
+ | mso-hansi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-font-family: | ||
+ | Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family:Wingdings">à</span></span><span lang="EN-US"> maybe I mixed up with the samples…? If so, all clones would contain | ||
+ | the GFP-Terminator plasmid. But this cannot be…<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings; | ||
+ | mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Sequencing of Re-trafo | ||
+ | clones 1, 4 and 5??? <span style="color:red">Ask Katrin G. about her opinion of | ||
+ | the gel!</span><o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Samples stored at -20°C in | ||
+ | “yellow-tip-box rag”</span></p> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US"><o:p> </o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Gel | ||
+ | run: Colony PCR for DarR and Riboswitch biobrick transformation from 15.7.13<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Addition of 5 µl 5xLD to the | ||
+ | samples</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Loading of 5 µl of each sample | ||
+ | on 1 % agarose gel</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Loading of 3 µl 2 log ladder as | ||
+ | a marker</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Gel run at 200 V</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">EtBr staining + destaining in | ||
+ | water</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">UV detection</span></p> | ||
+ | |||
+ | <p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class="MsoNormal"><span lang="EN-US">Gel 1:</span></p> | ||
+ | |||
+ | <p class="MsoNormal"><span lang="EN-US" style="mso-no-proof:yes"><!--[if gte vml 1]><v:shape | ||
+ | id="图片_x0020_2" o:spid="_x0000_i1026" type="#_x0000_t75" style='width:471pt; | ||
+ | height:366.75pt;visibility:visible;mso-wrap-style:square'> | ||
+ | <v:imagedata src="raw-07.18.files/image003.png" o:title="" croptop="-2039f" | ||
+ | cropbottom="-102f" cropleft="-826f" cropright="-1229f"/> | ||
+ | </v:shape><![endif]--><!--[if !vml]--><img width="500" src="https://static.igem.org/mediawiki/2013/thumb/6/62/Goe-18.07.13-RT-2.png/768px-Goe-18.07.13-RT-2.png" v:shapes="图片_x0020_2"><!--[endif]--></span></p> | ||
+ | |||
+ | <p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p> | ||
+ | |||
+ | <p class="MsoNormal"><span lang="EN-US">Gel 2:</span></p> | ||
+ | |||
+ | <p class="MsoNormal"><span lang="EN-US" style="mso-no-proof:yes"><!--[if gte vml 1]><v:shape | ||
+ | id="图片_x0020_3" o:spid="_x0000_i1025" type="#_x0000_t75" style='width:471pt; | ||
+ | height:420pt;visibility:visible;mso-wrap-style:square'> | ||
+ | <v:imagedata src="raw-07.18.files/image005.png" o:title="" croptop="-2410f" | ||
+ | cropbottom="-113f" cropleft="-1252f" cropright="-1303f"/> | ||
+ | </v:shape><![endif]--><!--[if !vml]--><img width="500" src="https://static.igem.org/mediawiki/2013/thumb/6/6d/Goe-18.07.13-RT-3.png/670px-Goe-18.07.13-RT-3.png" v:shapes="图片_x0020_3"><!--[endif]--></span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings; | ||
+ | mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><span lang="EN-US">Only for clone 4 two weak bands at 2 – 3 kb are seen, but this can | ||
+ | actually not correspond to any of the biobricks. Could be the plasmid</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l1 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:Wingdings;mso-fareast-font-family:Wingdings; | ||
+ | mso-bidi-font-family:Wingdings"><span style="mso-list:Ignore">è<span style="font:7.0pt "Times New Roman""> </span></span></span><!--[endif]--><span lang="EN-US">All clones are apparently negative corresponding to re-ligated | ||
+ | pSB1C3-terminator… Don’t forget gel ex next time…XD</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US">Samples stored at -20°C in | ||
+ | “yellow-tip-box rag”</span></p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="tlob" id="tl_0716"> | ||
+ | <span class="date">16th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
+ | <div class="cont"> | ||
+ | <p class="timeline-title goe-rt">Transformation from 15.7.13,Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10)</p> | ||
+ | <div class="timeline-cont"> | ||
+ | |||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Transformation | ||
+ | from 15.7.13</span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Amp-plates:<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">negative control | ||
+ | = negative/no clones</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">transformation | ||
+ | of RBS-GFP-Terminator C12 plasmid: many clones on 50 µl and on rest plate</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
+ | Calibri"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Cm-plates<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">Negative control | ||
+ | = negative</span></p> | ||
+ | |||
+ | <p class="MsoNormal" style="margin-left:36.0pt"><span lang="EN-US">Clones on all | ||
+ | other plates including w/o insert control (forgot gel extraction of pSB1C3 | ||
+ | vector </span><span lang="EN-US" style="font-family:Wingdings;mso-ascii-font-family: | ||
+ | "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-char-type:symbol; | ||
+ | mso-symbol-font-family:Wingdings"><span style="mso-char-type:symbol;mso-symbol-font-family: | ||
+ | Wingdings">à</span></span><span lang="EN-US"> terminator insert is still in | ||
+ | reaction mix; since we didn’t <span class="SpellE">dephosphorylate</span> the | ||
+ | vector with AP, there will be false positives corresponding to plasmid part | ||
+ | 7!); smaller and larger clones on all plates; further incubation over day since | ||
+ | they were too small to be picked in the morning…</span></p> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US"><o:p> </o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">Colony | ||
+ | PCR for RBS-GFP-Terminator C12 re-<span class="SpellE">trafo</span> clones 1 to | ||
+ | 10 (RT-C1 to RT-C10)<o:p></o:p></span></b></p> | ||
+ | |||
+ | <p class="MsoListParagraphCxSpFirst" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">According | ||
+ | to protocol from 10.7.13<o:p></o:p></span></p> | ||
+ | |||
+ | <p class="MsoListParagraphCxSpMiddle" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">16x | ||
+ | <span class="SpellE">MasterMix</span><o:p></o:p></span></p> | ||
+ | |||
+ | <p class="MsoListParagraphCxSpMiddle" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">Clones | ||
+ | 1 – 10 picked, streaked out on <span class="SpellE">LB<sup>Amp</sup></span>-plate | ||
+ | and used for inoculation of 25 µl reactions (blue tubes)<o:p></o:p></span></p> | ||
+ | |||
+ | <p class="MsoListParagraphCxSpLast" style="text-indent:-18.0pt;mso-list:l1 level1 lfo2"><!--[if !supportLists]--><span lang="EN-US" style="mso-bidi-font-family:Calibri;mso-ansi-language:EN-US"><span style="mso-list:Ignore">-<span style="font:7.0pt "Times New Roman""> | ||
+ | </span></span></span><!--[endif]--><span lang="EN-US" style="mso-ansi-language:EN-US">Controls: | ||
+ | 1 µl of 1:10 dilution of plasmids part 7, part 8 GFP-Terminator C1 and | ||
+ | RBS-GFP-Terminator C12 (yellow tubes)<o:p></o:p></span></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | <p class="timeline-title goe-dac">Concentration of dialysed protein samples<br />Determination of final protein concentration via Bradford test<br />SDS Page of dialysis and elution samples</p> | ||
+ | <div class="timeline-cont"> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt">Concentration | ||
+ | of dialysed protein samples</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Put E1 samples from dialysis in vivaspin | ||
+ | column</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Spin column for 20min at 4000xg</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Add 4ml of E2 to vivaspin and centrifuge | ||
+ | for 20min at 4000xg</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Resuspend and repeat centrifugation and | ||
+ | resuspension </span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt"> </span></b></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt"> </span></b></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt">Determination | ||
+ | of final protein concentration via Bradford test</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Add 1ml of Bradford solution (1:5 | ||
+ | dilution = 200ml Bradford + 800ml H<sub>2</sub>O) to 5µl of 1:20 diluted | ||
+ | protein solution</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation for 5min at RT</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Measure extinction at 595nm</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Calculation: c= OD<sub>595</sub> / | ||
+ | Vx0,0536</span></p> | ||
+ | <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none"> | ||
+ | <tbody><tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:12.0pt">Eluate</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:12.0pt">Absorption (nm)</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border:solid windowtext 1.0pt; | ||
+ | border-left:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="font-size:12.0pt">C [µg/ml]</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt">Sample 1</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt">0,121</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt">0,49</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="205" valign="top" style="width:153.5pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt">Sample 2</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt">0,123</span></p> | ||
+ | </td> | ||
+ | <td width="205" valign="top" style="width:153.55pt;border-top:none;border-left: | ||
+ | none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; | ||
+ | padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt">1,49</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="614" colspan="3" valign="top" style="width:460.6pt;border:solid windowtext 1.0pt; | ||
+ | border-top:none;padding:0cm 5.4pt 0cm 5.4pt"> | ||
+ | <p class="MsoNormal" align="right" style="text-align:right"><span lang="EN-US" style="font-size:12.0pt">Total: 9,35 mgml</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody></table> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt"> </span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:12.0pt">SDS Page of | ||
+ | dialysis and elution samples</span></b></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Mix 15µl of samples with 5µl Pab | ||
+ | and 5µl buffer W</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Incubation for 10min at 93°C </span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Mix each sample with 4µl protein marker</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Load whole volume on 15% SDS-gel</span></p> | ||
+ | <p class="MsoNormal" align="left" style="margin-left:36.0pt;text-align:left; | ||
+ | text-indent:-18.0pt;text-autospace:ideograph-other;vertical-align:baseline"><span lang="EN-US" style="font-size:12.0pt;font-family:"Arial","sans-serif"">-<span style="font:7.0pt "Times New Roman""> </span></span><span lang="EN-US" style="font-size:12.0pt">Run for 5min at 90V and 1h at 120V</span></p> | ||
+ | <p class="MsoNormal"><span lang="EN-US" style="font-size:12.0pt">D1 | D2 | D3 | M | ||
+ | | E1 | E2 |</span></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif""><img border="0" width="242" height="181" src="https://static.igem.org/mediawiki/2013/3/32/Goe-dac-Image018.jpg"></span></b></p> | ||
+ | <p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family: | ||
+ | "Arial","sans-serif""> </span></b></p> | ||
+ | |||
+ | |||
+ | <div class="fbutton">Fold ↑</div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
<div class="tlob" id="tl_0715"> | <div class="tlob" id="tl_0715"> | ||
<span class="date">15th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | <span class="date">15th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span> | ||
<div class="cont"> | <div class="cont"> | ||
- | <p class="timeline-title">Analysis of the RD samples from 12.7</p> | + | <p class="timeline-title goe-rt">Analysis of the RD samples from 12.7</p> |
<div class="timeline-cont"> | <div class="timeline-cont"> | ||
Line 1,667: | Line 2,014: | ||
</tr> | </tr> | ||
</tbody></table> | </tbody></table> | ||
+ | <br /> | ||
<div class="fbutton">Fold ↑</div> | <div class="fbutton">Fold ↑</div> |
Latest revision as of 20:04, 30 September 2013
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10), Colony PCR for DarR and Riboswitch biobrick transformation from 15.7.13
Gel
run: Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to
RT-C10)
- Addition of 5 µl 5xLD to the samples
- Loading of 5 µl of each sample on 1 % agarose gel
- Loading of 3 µl 2 log ladder as a marker
- Gel run at 100 V
- EtBr staining + destaining in water
- UV detection
Re-Trafo Clones 1, 4 – 7, 9 and 10 look promising: could contain RBS-GFP-Terminator construct in pSB1A3 backbone
è Re-Trafo Clone 2 has a strange additional band (from plasmid?)
è Re-Trafo Clone 8: no product
è Even for Plasmid of
RBS-GFP-Terminator C12, the additional band at the height of the terminator PCR
product is not seen à but for plasmid GFP this band could be visible… additionally strong
band of GFP-Terminator C1 PCR product runs higher than band of PCR product for
RBS-GFP-Terminator C12 à maybe I mixed up with the samples…? If so, all clones would contain
the GFP-Terminator plasmid. But this cannot be…
è Sequencing of Re-trafo
clones 1, 4 and 5??? Ask Katrin G. about her opinion of
the gel!
- Samples stored at -20°C in “yellow-tip-box rag”
Gel
run: Colony PCR for DarR and Riboswitch biobrick transformation from 15.7.13
- Addition of 5 µl 5xLD to the samples
- Loading of 5 µl of each sample on 1 % agarose gel
- Loading of 3 µl 2 log ladder as a marker
- Gel run at 200 V
- EtBr staining + destaining in water
- UV detection
Gel 1:
Gel 2:
è Only for clone 4 two weak bands at 2 – 3 kb are seen, but this can actually not correspond to any of the biobricks. Could be the plasmid
è All clones are apparently negative corresponding to re-ligated pSB1C3-terminator… Don’t forget gel ex next time…XD
- Samples stored at -20°C in “yellow-tip-box rag”
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Transformation from 15.7.13,Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10)
Transformation from 15.7.13
-
Amp-plates:
negative control = negative/no clones
transformation of RBS-GFP-Terminator C12 plasmid: many clones on 50 µl and on rest plate
-
Cm-plates
Negative control = negative
Clones on all other plates including w/o insert control (forgot gel extraction of pSB1C3 vector à terminator insert is still in reaction mix; since we didn’t dephosphorylate the vector with AP, there will be false positives corresponding to plasmid part 7!); smaller and larger clones on all plates; further incubation over day since they were too small to be picked in the morning…
Colony
PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to
10 (RT-C1 to RT-C10)
-
According
to protocol from 10.7.13
-
16x
MasterMix
-
Clones
1 – 10 picked, streaked out on LBAmp-plate
and used for inoculation of 25 µl reactions (blue tubes)
-
Controls:
1 µl of 1:10 dilution of plasmids part 7, part 8 GFP-Terminator C1 and
RBS-GFP-Terminator C12 (yellow tubes)
Concentration of dialysed protein samples
Determination of final protein concentration via Bradford test
SDS Page of dialysis and elution samples
Concentration of dialysed protein samples
- Put E1 samples from dialysis in vivaspin column
- Spin column for 20min at 4000xg
- Add 4ml of E2 to vivaspin and centrifuge for 20min at 4000xg
- Resuspend and repeat centrifugation and resuspension
Determination of final protein concentration via Bradford test
- Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H2O) to 5µl of 1:20 diluted protein solution
- Incubation for 5min at RT
- Measure extinction at 595nm
- Calculation: c= OD595 / Vx0,0536
Eluate |
Absorption (nm) |
C [µg/ml] |
Sample 1 |
0,121 |
0,49 |
Sample 2 |
0,123 |
1,49 |
Total: 9,35 mgml |
SDS Page of dialysis and elution samples
- Mix 15µl of samples with 5µl Pab and 5µl buffer W
- Incubation for 10min at 93°C
- Mix each sample with 4µl protein marker
- Load whole volume on 15% SDS-gel
- Run for 5min at 90V and 1h at 120V
D1 | D2 | D3 | M | E1 | E2 |
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Analysis of the RD samples from 12.7
Test run of plasmids purified on 12.7.13, vector and inserts on agarose gel
- part 7 plasmid, purified on 12.7.13
- pSB1C3 vector from part 7, purified on 11.7.13
- plasmid RBS-GFP-Terminator C12
- plasmid RBS-GFP-Terminator C12 test restriction with EcoRI
- inserts of DarR/riboswitches after 2nd digest with EcoRI
- 1% agarose-1xTAE-gel
- 3 µl 2 log ladder loaded on both sides of gel as a marker
Samples:
Component |
Part
7 plasmid (179.3
ng/µl) |
pSB1C3 (2nd
elution, 3.4 ng/µl) |
plasmid
RBS-GFP-Terminator C12 (200.8
ng/µl) |
plasmid
RBS-GFP-Terminator C12 R.D. |
DarR
purified PCR product (4 µl primer; reaction from 24.6.13; 15.2 ng/µl) |
DarR
insert |
DNA |
0.5 µl |
4 µl |
0.5 µl |
5 µl |
2 µl |
3 µl |
dH2O |
3.5 µl |
- |
3.5 µl |
- |
2 µl |
1 µl |
5xLD |
1 µl |
1 µl |
1 µl |
-(sample in green FD buffer |
1 µl |
1 µl |
Component |
Riboswitch
iGEM_40/41 Purified
PCR product (45.7
ng/µl) |
Riboswitch
iGEM_40/41 insert |
Riboswitch
iGEM_41/42 Purified
PCR product (36.8
ng/µl) |
Riboswitch
iGEM_41/42 insert |
Riboswitch
iGEM_40/43 Purified
PCR product (47.7
ng/µl) |
Riboswitch
iGEM_40/43 insert |
Riboswitch
iGEM_42/43 Purified
PCR product (35.7
ng/µl) |
Riboswitch
iGEM_42/43 insert |
DNA |
1 µl |
3 µl |
- |
3 µl |
1 µl |
3 µl |
- |
3 µl |
dH2O |
3 µl |
1 µl |
4 µl |
1 µl |
3 µl |
1 µl |
4 µl |
1 µl |
5xLD |
1 µl |
1 µl |
1 µl |
1 µl |
1 µl |
1 µl |
1 µl |
1 µl |
(for Riboswitch iGEM_41/42 and _42/43, there were too small amounts of PCR product left -> control was not possible…)
- Run at 100 V
- EtBr staining + destaining in water
- UV detection
![](https://static.igem.org/mediawiki/2013/6/60/Goe-15.07.13-RT.png)
Purification of DarR and Riboswitch inserts
- Qiagen PCR purification kit
- 500 µl PB used for each reaction
- 2x elution in pre-warmed HPLC water (25 µl)
- NanoDrop concentration measurements (for some samples, the concentration was measured twice, since first values for 1st and 2nd elution were a little strange… -> approximate average concentration can be used for further calculations)
Sample |
Concentration (ng/µl) |
Average concentration |
A260/A280 |
A260/A230 |
Riboswitch iGEM_40/41 insert |
7.5 |
1.74 |
1.01 |
|
Riboswitch iGEM_40/43 insert |
5.5 |
1.18 |
1.09 |
|
Riboswitch iGEM_42/41 insert |
2.3 |
3 |
1.89 |
0.86 |
4.6 |
1.38 |
0.76 |
||
Riboswitch iGEM_42/43 insert |
3.0 |
3 |
2.18 |
0.76 |
3.1 |
1.09 |
0.90 |
||
DarR insert |
1.7 |
2 |
3.34 |
0.71 |
2.4 |
1.06 |
0.62 |
2nd elution
Sample |
Concentration (ng/µl) |
Average concentration |
A260/A280 |
A260/A230 |
Riboswitch iGEM_40/41 insert |
5.2 |
1.30 |
0.56 |
|
Riboswitch iGEM_40/43 insert |
1.5 |
0.99 |
0.36 |
|
Riboswitch iGEM_42/41 insert |
2.6 |
3 |
1.38 |
0.52 |
3.6 |
1.18 |
0.62 |
||
Riboswitch iGEM_42/43 insert |
3.9 |
4 |
1.14 |
0.63 |
4.2 |
1.07 |
0.61 |
||
DarR insert |
2.9 |
1.04 |
0.52 |