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Tokyo-Nokogen-Notebook
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This chart suggests that we pick up part 1 and part 2 materials, ligate them, transform DH5a with them, inoculate that cell and do miniprep after cultivation. 【red light receptor】 Construct parts <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Order primers which designed to amplify antigen43. <p style="text-indent: 3em">Construct parts. <td style="background-color:#666; color:#FFF" nowrap>

<p style="text-indent: 2em">★ 08/24/09 to 08/28/09 ---parts construction <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Construct parts <p style="text-indent: 3em">【red light receptor】 <p style="text-indent: 3em">Construct parts <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Extract genome contains antigen43 from E.coli K-12. <p style="text-indent: 3em">Amplify antigen43 by PCR using primers we made. <p style="text-indent: 3em">Construct parts

<p style="text-indent: 2em">★ 08/31/09 to 09/04/09 -parts construction <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Construct parts. <p style="text-indent: 3em">Make shortage plates and LB medium. <p style="text-indent: 3em">【red light receptor】 <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Check sequence of [a1] and [a2], but cannot read it. <p style="text-indent: 3em">Gel electrophoresis of antigen43 and expression vector pTrc99- Nde Ⅰ cut by Nde Ⅰ, <p style="text-indent: 3em">but cannot find appropriate bands. <td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 09/07/09 to 09/11/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">【green light receptor】 <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Gel electrophoresis of antigen43 cut by Nde Ⅰand Spe Ⅰ. <p style="text-indent: 3em">Gel electrophoresis of expression vector pTrc99- Nde Ⅰ cut by Nde Ⅰand Xba Ⅰ <p style="text-indent: 3em">Do geneclean both of them, ligate them, transform DH5a with ligated sample and inoculate it. <p style="text-indent: 3em">Check sequence of [a1] and [a2] again, but cannot read it. <p style="text-indent: 2em">★ 09/14/09 to 09/18/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Sequence analysis. <p style="text-indent: 3em">【green light receptor】 <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em"> Pick up some single colonies from inoculated plate made last week and do direct colony PCR. After PCR, <p style="text-indent: 3em">we check presence of insert DNA in plasmid by electrophoresis but cannot find. <p style="text-indent: 3em">Order primers which break 6 Pst Ⅰsites not to change amino acid sequence. <p style="text-indent: 3em">Try reconstruction of [a1] to [a3]. <td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 09/21/09 to 09/25/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">From results of sequence analysis, we find we couldn’t construct all of the above counter parts. <p style="text-indent: 3em">And start reconstruction of counter parts ([c1], [c2] [c4] and following parts.) <p style="text-indent: 3em">【green light receptor】 <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Gel electrophoresis of antigen43 cut by Nde Ⅰand Spe Ⅰ. <p style="text-indent: 3em">Gel electrophoresis of expression vector pTrc99- Nde Ⅰ cut by Nde Ⅰand Xba Ⅰ <p style="text-indent: 3em">Do geneclean both of them, ligate them, transform DH5a with ligated sample and inoculate it. <p style="text-indent: 3em">Try reconstruction of [a4] and [a5]. <p style="text-indent: 2em">★ 09/21/09 to 09/25/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Reconstruction of parts ([c3], [c6], [c12] and follwing part) <p style="text-indent: 3em">Check sequence of reconstruction parts. <p style="text-indent: 3em">【light receptor】 <p style="text-indent: 3em">Test red light receptor. <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Pick up some single colonies from inoculated plate made last week and do direct colony PCR. <p style="text-indent: 3em">After PCR, we check presence of insert DNA in plasmid by electrophoresis but cannot find. <p style="text-indent: 3em">Pick up all colonies and cultivate them and do induction in order to watch out occurrence of aggregation. <p style="text-indent: 3em">But we cannot see aggregation.

<td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 09/28/09 to 10/02/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Reconstruction of parts ([c5] and following parts) <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【light receptor】 <p style="text-indent: 3em">Test green light receptor. <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Try TA cloning (ligation of antigen43 with pGEMT vector). <p style="text-indent: 3em">Transform DH5a with ligated sample, inoculate it, cultivate it and do miniprep. <p style="text-indent: 3em">Try mutation 1st and 2nd PstⅠ sites. After mutagenesis, transform DH5a with ligated sample, inoculate it. <p style="text-indent: 3em">But there is no colony on plate.

<p style="text-indent: 2em">★ 10/5/09 to 10/09/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Check sequence of [a1] and [a2] again, but cannot read full length sequence. <p style="text-indent: 3em">→ Something wrong with Biobrick parts.

<td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 10/12/09 to 10/16/09 <p style="text-indent: 3em">Start testing all parts. <p style="text-indent: 3em">Prepare for sending parts to MIT  <img src="http://2009.igem.org/wiki/images/9/90/TOP4.png" width="63" height="50" border="0"></a>

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