Difference between revisions of "Team:Munich/chibiobrick2.html"

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<div class="event-info">
 
<div class="event-info">
  
−
<h3>7/31</h3>
+
<h4>Forming oligodimers from single-strand DNA chi6 sequences</h4>
−
<h4>Chi6 Oligodimerization</h4>
+
<em>2018/07/31</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
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</table>
 
</table>
  
−
 
+
<h4>Assembling pSB1C3_Chi6 with A3 Assembly</h4>
−
<h3>8/1</h3>
+
<em>2018/08/01</em>
−
<h4>assembling pSB1C3_Chi6</h4>
+
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
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<tr>
 
<tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>XbaI, SpeI-HF; BB: 46 ng/µl
+
       <td>Restriction digest with XbaI, SpeI-HF
−
</td>
+
−
    </tr>
+
−
<tr>
+
−
      <td>Results:</td>
+
−
      <td></td>
+
−
    </tr>
+
−
</table>
+
−
 
+
−
<h3>8/2</h3>
+
−
<h4>PCR to assemble Chi6 oligomers</h4>
+
−
<table class="table table-borderless">
+
−
    <tr>
+
−
 
+
−
      <td>Participants:</td>
+
−
      <td>Enikö</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Protocol:</td>
+
−
      <td>PCR</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Notes:</td>
+
−
      <td>
+
−
</td>
+
−
    </tr>
+
−
<tr>
+
−
      <td>Results:</td>
+
−
      <td></td>
+
−
    </tr>
+
−
</table>
+
−
 
+
−
<h3>8/3</h3>
+
−
<h4>Gibson assembly to make pSB1C3_Chi6</h4>
+
−
<table class="table table-borderless">
+
−
    <tr>
+
−
 
+
−
      <td>Participants:</td>
+
−
      <td>Enikö</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Protocol:</td>
+
−
      <td>Gibson assembly</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Notes:</td>
+
−
      <td>
+
−
</td>
+
−
    </tr>
+
−
<tr>
+
−
      <td>Results:</td>
+
−
      <td></td>
+
−
    </tr>
+
−
</table>
+
−
 
+
−
<h3>8/3</h3>
+
−
<h4>Ligation to make pSB1C3_Chi6</h4>
+
−
<table class="table table-borderless">
+
−
    <tr>
+
−
 
+
−
      <td>Participants:</td>
+
−
      <td>Enikö</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Protocol:</td>
+
−
      <td>Ligation</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Notes:</td>
+
−
      <td>
+
−
</td>
+
−
    </tr>
+
−
<tr>
+
−
      <td>Results:</td>
+
−
      <td></td>
+
−
    </tr>
+
−
</table>
+
−
 
+
−
<h3>8/3</h3>
+
−
<h4>transforming DH5a with pSB1C3_Chi6</h4>
+
−
<table class="table table-borderless">
+
−
    <tr>
+
−
 
+
−
      <td>Participants:</td>
+
−
      <td>Enikö</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Protocol:</td>
+
−
      <td>chem trafo</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Notes:</td>
+
−
      <td>
+
 
</td>
 
</td>
 
     </tr>
 
     </tr>
Line 132: Line 39:
 
</table>
 
</table>
  
−
<h3>8/6</h3>
+
<h4>Redo: Assembling pSB1C3_Chi6 with Ligation</h4>
−
<h4>DNA Mini prep from E. Coli Dh5a pSB1C3_Chi6</h4>
+
<em>2018/08/02 - 2018/08/06</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
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     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>mini prep</td>
+
       <td>PCR, PCR purify, gibson assembly, ligation, chem trafo, mini-prep, agarose gel</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>
+
       <td>We performed linear amplification of Chi6 single stranded DNA because assembly failed before. gibson assembly and ligation was performed in parallel<br>
 +
Primer: BBS-PstI-rv
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
 
       <td>Results:</td>
 
       <td>Results:</td>
−
       <td>concentration</td>
+
       <td>Colonies were obtained but consequent test-pcr showed no correct bands on the gel. The colonies had to be contaminants. Eni suspected errors during the PCR reaction.</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
−
<h3>8/6</h3>
+
<h4>Redo: Assembling pSB1C3_Chi6 with Ligation</h4>
−
<h4>Test Digest of pSB1C3_Chi6</h4>
+
<em>2018/08/07</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
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     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>Restrictiondigest</td>
+
       <td>PCR, agarose gel</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>
+
       <td>Primer: BBS-PstI-rv
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
 
       <td>Results:</td>
 
       <td>Results:</td>
−
       <td>no bands</td>
+
       <td>no bands on the gel. Indeed, the error before occured during the pcr reaction. Thomas (supervisor) and Eni decided to give A3 assembly one more try.</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
−
<h3>8/7</h3>
+
<h4>Assembling pSB1C3_Chi6 with A3 assembly</h4>
−
<h4>PCR to create Chi6 dimer</h4>
+
<em>2018/08/07 - 2018/08/09</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
Line 188: Line 96:
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>PCR</td>
+
       <td>Restrictiondigest, Pcr purify, ligation, chem trafo, mini-prep, sequencing</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>
+
       <td>We switched to the enzymes EcoRI, SpeI because we realized XbaI and SpeI had the same cutting sites and might lead to religation or false direction.
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
 
       <td>Results:</td>
 
       <td>Results:</td>
−
       <td>no bands</td>
+
       <td>We obtained colonies, however repeated sequencing with VF2 and VR didnt give successful reads.</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
−
<h3>8/7</h3>
 
−
<h4>Digest Chi6 with EcoRI, SpeI</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Enikö</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>Restrictiondigest</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>
 
−
</td>
 
−
    </tr>
 
−
<tr>
 
−
      <td>Results:</td>
 
−
      <td>no bands</td>
 
−
    </tr>
 
−
</table>
 
−
 
−
<h3>8/7</h3>
 
−
<h4>PCR purify Chi6 digested with EcoRI, SpeI</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Enikö</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>PCR purify</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>
 
−
</td>
 
−
    </tr>
 
−
<tr>
 
−
      <td>Results:</td>
 
−
      <td></td>
 
−
    </tr>
 
−
</table>
 
−
 
−
<h3>8/7</h3>
 
−
<h4>Ligate pSB1C3 and Chi6 digested with EcoRI, SpeI</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Enikö</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>Ligation</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>
 
−
</td>
 
−
    </tr>
 
−
<tr>
 
−
      <td>Results:</td>
 
−
      <td>colonies</td>
 
−
    </tr>
 
−
</table>
 
−
 
−
<h3>8/8</h3>
 
−
<h4>DNA miniprep pSB1C3_Chi6</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Enikö</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>miniprep</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>
 
−
</td>
 
−
    </tr>
 
−
<tr>
 
−
      <td>Results:</td>
 
−
      <td>concentration</td>
 
−
    </tr>
 
−
</table>
 
−
 
−
<h3>8/8</h3>
 
−
<h4>sequencing pSB1C3_Chi6</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Enikö</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>sequencing</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>
 
−
</td>
 
−
    </tr>
 
−
<tr>
 
−
      <td>Results:</td>
 
−
      <td>?</td>
 
−
    </tr>
 
−
</table>
 
−
 
−
<h3>8/9</h3>
 
−
<h4>sequencing pSB1C3_Chi6</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Enikö</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>sequencing</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>
 
−
</td>
 
−
    </tr>
 
−
<tr>
 
−
      <td>Results:</td>
 
−
      <td>?</td>
 
−
    </tr>
 
−
</table>
 
−
 
−
<h3>8/12</h3>
 
 
<h4>PCR to assemble pSB1C3_Chi6</h4>
 
<h4>PCR to assemble pSB1C3_Chi6</h4>
 +
<em>2018/08/16 - 2018/08/20</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
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     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>PCR</td>
+
       <td>PCR, restriction digest, pcr purify, ligation, chem trafo</td>
−
    </tr>
+
−
    <tr>
+
−
      <td>Notes:</td>
+
−
      <td>expected: 2kb
+
−
</td>
+
−
    </tr>
+
−
<tr>
+
−
      <td>Results:</td>
+
−
      <td>?</td>
+
−
    </tr>
+
−
</table>
+
−
 
+
−
<h3>8/16</h3>
+
−
<h4>PCR to assemble pSB1C3_Chi6</h4>
+
−
<table class="table table-borderless">
+
−
    <tr>
+
−
 
+
−
      <td>Participants:</td>
+
−
      <td>Enikö</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Protocol:</td>
+
−
      <td>PCR</td>
+
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
 
       <td>because oligodimerisation & ligation failed, we decided to amplify chi6 by pcr; primers: Chi6_2clone & Chi6_fill <br>
 
       <td>because oligodimerisation & ligation failed, we decided to amplify chi6 by pcr; primers: Chi6_2clone & Chi6_fill <br>
−
cycle: 98° 2 min; 98° 20 s; 57° 30s; 72° 20s (to 2 5 x) 72° 2 min <br>
+
cycle: 98° 2 min; 98° 20 s; 57° 30s; 72° 20s (to 2 5 x) 72° 2 min;
 
expect: ca 100 bp <br>
 
expect: ca 100 bp <br>
 
Backbone: BBS_AgeI_chi6_fw & BBP_SalI_Chi8_rv; AT: 62° expect: 2 kb <br>
 
Backbone: BBS_AgeI_chi6_fw & BBP_SalI_Chi8_rv; AT: 62° expect: 2 kb <br>
 +
next, we digested the samples with ageI, SalI
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
 
       <td>Results:</td>
 
       <td>Results:</td>
−
       <td>?</td>
+
       <td>Didnt work, Eni ordered 3 different primer pairs to stitch chi6 together by pcr. </td>
−
    </tr>
+
−
</table>
+
−
 
+
−
<h3>8/20</h3>
+
−
<h4>digest Chi6 to assemble pSB1C3_chi6</h4>
+
−
<table class="table table-borderless">
+
−
    <tr>
+
−
 
+
−
      <td>Participants:</td>
+
−
      <td>Enikö</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Protocol:</td>
+
−
      <td>restrictiondigest</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td>Notes:</td>
+
−
      <td>ageI, SalI
+
−
</td>
+
−
    </tr>
+
−
<tr>
+
−
      <td>Results:</td>
+
−
      <td>didnt work, ordered 3 different primer pairs </td>
+
 
     </tr>
 
     </tr>
 
</table>
 
</table>
  
−
<h3>8/30</h3>
 
 
<h4>preparing pSB1C3 backbone</h4>
 
<h4>preparing pSB1C3 backbone</h4>
 +
<em>2018/08/30</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
Line 434: Line 159:
 
</table>
 
</table>
  
−
<h3>8/30</h3>
+
<h4>dimerization of short chi primers to assemble pSB1C3_chi6</h4>
−
<h4>dimerization of chi primers to assemble pSB1C3_chi6</h4>
+
<em>2018/08/30 - 2018/09/05</em>
 
<table class="table table-borderless">
 
<table class="table table-borderless">
 
     <tr>
 
     <tr>
Line 444: Line 169:
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>olidimerization, Agarose Gel, Gel extraction, Ligation, chem trafo</td>
+
       <td>oligodimerization, Agarose Gel, Gel extraction, Ligation, chem trafo, PCR, mini-prep</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Notes:</td>
 
       <td>Notes:</td>
−
       <td>
+
       <td>the primers were stitched together and purified from agarose gel. The samples were ligated into pSB1C3 and transformed into E.Coli NEB Turbo.
−
</td>
+
The samples were tested by test-pcr via the primers VF2 and VR.
−
    </tr>
+
−
<tr>
+
−
      <td>Results:</td>
+
−
      <td>colonies</td>
+
−
    </tr>
+
−
</table>
+
  
−
 
−
<h3>9/2</h3>
 
−
<h4>Test-PCR of pSB1C3_chi6</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Enikö</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>PCR</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>VF2, VR; expected: 414bp
 
 
</td>
 
</td>
 
     </tr>
 
     </tr>
 
  <tr>
 
  <tr>
 
       <td>Results:</td>
 
       <td>Results:</td>
−
       <td>colonies</td>
+
       <td>colonies. consequent test-pcr proved the correctness of pSB1C3_Chi6. (PIC)</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
−
 
−
<h3>9/5</h3>
 
−
<h4>Mini-prep of pSB1C3_chi6</h4>
 
−
<table class="table table-borderless">
 
−
    <tr>
 
−
 
−
      <td>Participants:</td>
 
−
      <td>Katja</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Protocol:</td>
 
−
      <td>mini prep</td>
 
−
    </tr>
 
−
    <tr>
 
−
      <td>Notes:</td>
 
−
      <td>
 
−
</td>
 
−
    </tr>
 
−
<tr>
 
−
      <td>Results:</td>
 
−
      <td></td>
 
−
    </tr>
 
−
</table>
 
−
 
  
 
</div>
 
</div>
 
</html>
 
</html>

Revision as of 13:18, 29 September 2018

Forming oligodimers from single-strand DNA chi6 sequences

2018/07/31
Participants: Domi
Protocol: Oligodimerization

Assembling pSB1C3_Chi6 with A3 Assembly

2018/08/01
Participants: Domi
Protocol: Restriction digest, PCR purify, Gibson assembly, chem.trafo
Notes: Restriction digest with XbaI, SpeI-HF
Results: no colonies

Redo: Assembling pSB1C3_Chi6 with Ligation

2018/08/02 - 2018/08/06
Participants: Enikö
Protocol: PCR, PCR purify, gibson assembly, ligation, chem trafo, mini-prep, agarose gel
Notes: We performed linear amplification of Chi6 single stranded DNA because assembly failed before. gibson assembly and ligation was performed in parallel
Primer: BBS-PstI-rv
Results: Colonies were obtained but consequent test-pcr showed no correct bands on the gel. The colonies had to be contaminants. Eni suspected errors during the PCR reaction.

Redo: Assembling pSB1C3_Chi6 with Ligation

2018/08/07
Participants: Enikö
Protocol: PCR, agarose gel
Notes: Primer: BBS-PstI-rv
Results: no bands on the gel. Indeed, the error before occured during the pcr reaction. Thomas (supervisor) and Eni decided to give A3 assembly one more try.

Assembling pSB1C3_Chi6 with A3 assembly

2018/08/07 - 2018/08/09
Participants: Enikö
Protocol: Restrictiondigest, Pcr purify, ligation, chem trafo, mini-prep, sequencing
Notes: We switched to the enzymes EcoRI, SpeI because we realized XbaI and SpeI had the same cutting sites and might lead to religation or false direction.
Results: We obtained colonies, however repeated sequencing with VF2 and VR didnt give successful reads.

PCR to assemble pSB1C3_Chi6

2018/08/16 - 2018/08/20
Participants: Enikö
Protocol: PCR, restriction digest, pcr purify, ligation, chem trafo
Notes: because oligodimerisation & ligation failed, we decided to amplify chi6 by pcr; primers: Chi6_2clone & Chi6_fill
cycle: 98° 2 min; 98° 20 s; 57° 30s; 72° 20s (to 2 5 x) 72° 2 min; expect: ca 100 bp
Backbone: BBS_AgeI_chi6_fw & BBP_SalI_Chi8_rv; AT: 62° expect: 2 kb
next, we digested the samples with ageI, SalI
Results: Didnt work, Eni ordered 3 different primer pairs to stitch chi6 together by pcr.

preparing pSB1C3 backbone

2018/08/30
Participants: Enikö
Protocol: Restriction digest, Agarose gel, gelextraction
Notes: EcoRi & SpeI
Results: PIC

dimerization of short chi primers to assemble pSB1C3_chi6

2018/08/30 - 2018/09/05
Participants: Enikö
Protocol: oligodimerization, Agarose Gel, Gel extraction, Ligation, chem trafo, PCR, mini-prep
Notes: the primers were stitched together and purified from agarose gel. The samples were ligated into pSB1C3 and transformed into E.Coli NEB Turbo. The samples were tested by test-pcr via the primers VF2 and VR.
Results: colonies. consequent test-pcr proved the correctness of pSB1C3_Chi6. (PIC)