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

Line 8: Line 8:
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Domi</td>
+
       <td>Dominic Schwarz</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>Oligodimerization</td>
+
       <td>
 +
<a href="https://www.sigmaaldrich.com/technical-documents/protocols/biology/annealing-oligos.html" target="_blank">Oligodimerization</a>
 +
</td>
 
     </tr>
 
     </tr>
 
</table>
 
</table>
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       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Domi</td>
+
       <td>Dominic Schwarz</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>Restriction digest, PCR purify, Gibson assembly, chem.trafo</td>
+
       <td>
 +
<a href="https://static.igem.org/mediawiki/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restrition digest</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,  
 +
<a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson assembly</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical Transformation</a>
 +
</td>
 
     </tr>
 
     </tr>
 
<tr>
 
<tr>
Line 45: Line 52:
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Enikö</td>
+
       <td>Enikö Baliács</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>PCR, PCR purify, gibson assembly, ligation, chem trafo, mini-prep, agarose gel</td>
+
       <td>
 +
<a href="https://static.igem.org/mediawiki/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,  
 +
<a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson assembly</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Mini prep</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>
 +
</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
Line 69: Line 84:
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Enikö</td>
+
       <td>Enikö Baligács</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>PCR, agarose gel</td>
+
       <td>
 +
<a href="https://static.igem.org/mediawiki/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a>,
 +
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>
 +
PCR, agarose gel</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
Line 92: Line 110:
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Enikö</td>
+
       <td>Enikö Baligács</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>Restrictiondigest, Pcr purify, ligation, chem trafo, mini-prep, sequencing</td>
+
       <td>
 +
<a href="https://static.igem.org/mediawiki/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restrition digest</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Mini Prep</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/a/ab/T--Munich--WL1_Sequencing_sample_preparation.pdf" target="_blank">Sequncing</a></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
Line 115: Line 139:
  
 
       <td>Participants:</td>
 
       <td>Participants:</td>
−
       <td>Enikö</td>
+
       <td>Enikö Baligács</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
 
       <td>Protocol:</td>
 
       <td>Protocol:</td>
−
       <td>PCR, restriction digest, pcr purify, ligation, chem trafo</td>
+
       <td>
 +
<a href="https://static.igem.org/mediawiki/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restrition digest</a>,  
 +
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>,  
 +
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a>
 +
</td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>

Revision as of 15:29, 15 October 2018

Forming oligodimers from single-strand DNA chi6 sequences

2018/07/31
Participants: Dominic Schwarz
Protocol: Oligodimerization

Assembling pSB1C3_Chi6 with A3 Assembly

2018/08/01
Participants: Dominic Schwarz
Protocol: Restrition digest, PCR purification, Gibson assembly, Chemical Transformation
Notes: Restriction digest with XbaI, SpeI-HF
Results: no colonies

Redo: Assembling pSB1C3_Chi6 with Ligation

2018/08/02 - 2018/08/06
Participants: Enikö Baliács
Protocol: PCR, PCR purification, Gibson assembly, Ligation, Chemical transformation, 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 Baligács
Protocol: PCR, Agarose gel 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 Baligács
Protocol: Restrition digest, PCR purification, Ligation, Chemical transformation, Mini Prep, Sequncing
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 Baligács
Protocol: PCR, Restrition digest, PCR purification, Ligation, Chemical transformation
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)