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

Revision as of 19:06, 16 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:	Restriction 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, Miniprep, 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
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:	Restriction 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, Restriction 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 Baligács
Protocol:	Restriction digest, Agarose gel, Gel extraction
Notes:	EcoRi & SpeI
Results:	PIC

dimerization of short chi primers to assemble pSB1C3_chi6

2018/08/30 - 2018/09/05

Participants:	Enikö Baligács
Protocol:	Oligodimerization, Agarose gel, Gel extraction, Ligation, Chemical Transformation, 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)

@@ Line 42: / Line 42: @@
   <tr>
        <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
-       <td>no colonies</td>
+       <td>No colonies</td>
      </tr>
 </table>
@@ Line 59: / Line 59: @@
 <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://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://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a>,
 <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>
 </td>
@@ Line 68: / Line 68: @@
      <tr>
        <td>Notes:</td>
-       <td>We performed linear amplification of Chi6 single stranded DNA because assembly failed before. gibson assembly and ligation was performed in parallel<br>
+       <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>
@@ Line 74: / Line 74: @@
   <tr>
        <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></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>
+       <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>
 </table>
@@ Line 100: / Line 100: @@
   <tr>
        <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></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>
+       <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>
 </table>
@@ Line 153: / Line 153: @@
      <tr>
        <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;
 expect: ca 100 bp <br>
 Backbone: BBS_AgeI_chi6_fw & BBP_SalI_Chi8_rv; AT: 62° expect: 2 kb <br>
-next, we digested the samples with ageI, SalI
+Next, we digested the samples with ageI, SalI
 </td>
      </tr>
   <tr>
        <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
-       <td>Didnt work, Eni ordered 3 different primer pairs to stitch chi6 together by pcr. </td>
+       <td>Didnt work, Eni ordered 3 different primer pairs to stitch chi6 together by PCR. </td>
      </tr>
 </table>
@@ Line 215: / Line 215: @@
      <tr>
        <td>Notes:</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 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.
+The samples were tested by test-PCR via the primers VF2 and VR.
 </td>
@@ Line 222: / Line 222: @@
   <tr>
        <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td>
-       <td>colonies. consequent test-pcr proved the correctness of pSB1C3_Chi6. (PIC)</td>
+       <td>Colonies. Consequent test-PCR proved the correctness of pSB1C3_Chi6. (PIC)</td>
      </tr>
 </table>