Difference between revisions of "Team:Munich/recbcdwt.html"
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| − | <h4>Assembling pSB1C3_RecBCD(WT) with | + | <h4>Assembling pSB1C3_RecBCD(WT) with Gibson Assembly</h4> |
<em>2018/08/06</em> | <em>2018/08/06</em> | ||
<table class="table table-borderless"> | <table class="table table-borderless"> | ||
| Line 16: | Line 16: | ||
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | ||
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | ||
| − | <a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson | + | <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> | <a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a> | ||
</td> | </td> | ||
| Line 51: | Line 51: | ||
</table> | </table> | ||
| − | <h4>redo: Assembling pSB1C3_RecBCD(WT) with | + | <h4>redo: Assembling pSB1C3_RecBCD(WT) with Gibson Assembly</h4> |
<em>2018/08/08</em> | <em>2018/08/08</em> | ||
<table class="table table-borderless"> | <table class="table table-borderless"> | ||
| Line 65: | Line 65: | ||
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | ||
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | ||
| − | <a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson | + | <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> | <a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a> | ||
</td> | </td> | ||
| Line 109: | Line 109: | ||
<td>Protocol:</td> | <td>Protocol:</td> | ||
<td> | <td> | ||
| − | <a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank"> | + | <a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a> |
</td> | </td> | ||
</tr> | </tr> | ||
| Line 126: | Line 126: | ||
<td> | <td> | ||
<a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</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/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson | + | <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/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a> | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a> | ||
</td> | </td> | ||
| Line 132: | Line 132: | ||
<tr> | <tr> | ||
<td>Notes:</td> | <td>Notes:</td> | ||
| − | <td> | + | <td> To improve cell survival we added 1% glucose to lb media |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td> | <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td> | ||
| − | <td> | + | <td>No colonies?</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 166: | Line 166: | ||
</table> | </table> | ||
| − | <h4>redo: Assembling pSB1C3_RecBCD(WT) with | + | <h4>redo: Assembling pSB1C3_RecBCD(WT) with Gibson Assembly</h4> |
<em>2018/08/14</em> | <em>2018/08/14</em> | ||
<table class="table table-borderless"> | <table class="table table-borderless"> | ||
| Line 180: | Line 180: | ||
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | ||
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | ||
| − | <a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson | + | <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> | <a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a> | ||
</td> | </td> | ||
| Line 236: | Line 236: | ||
<tr> | <tr> | ||
<td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td> | <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td> | ||
| − | <td> | + | <td>no results</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| − | <h4>Assembling pSB1C3_RecBCD(WT) with | + | <h4>Assembling pSB1C3_RecBCD(WT) with Gibson Assembly</h4> |
<em>2018/08/27</em> | <em>2018/08/27</em> | ||
<table class="table table-borderless"> | <table class="table table-borderless"> | ||
| Line 254: | Line 254: | ||
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | ||
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | ||
| − | <a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson | + | <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> | <a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a> | ||
</td> | </td> | ||
| Line 309: | Line 309: | ||
<a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | ||
<a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">Gel purification</a>, | ||
| − | <a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson | + | <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> | <a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical transformation</a> | ||
</td> | </td> | ||
| Line 321: | Line 321: | ||
<tr> | <tr> | ||
<td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td> | <td><a href="https://2018.igem.org/Team:Munich/Results" target="_blank">Results:</a></td> | ||
| − | <td> | + | <td>No colonies for pSB1C3_RecBCD-His</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| Line 336: | Line 336: | ||
<td>Protocol:</td> | <td>Protocol:</td> | ||
<td> | <td> | ||
| − | <a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank"> | + | <a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Miniprep</a>, |
<a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Sequencing</a>, | <a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Sequencing</a>, | ||
<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/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a> | ||
Revision as of 19:40, 16 October 2018
Assembling pSB1C3_RecBCD(WT) with Gibson Assembly
2018/08/06| Participants: | Enikö Baligács, Katja Neishsalo |
| Protocol: | PCR, Agarose gel, Gel purification, Gibson Assembly, Chemical transformation |
| Notes: |
BB & LacO
Primer: GA_lacO_rv & GA_lact_fw
expect: 2,5 kb RecC-UTR for GA GA_RecC_fw & GA_RecB_rv expect: 3,5 kb RecB/D for GA GA_RecB_fw & GA_RecD_rv expect: 5,3 kb; repeated because didnt work BB & LacO for GG SapI Lact_His_SapI_fw & RecC_lacO_His_SapI_rv expect: 2,5 kb RecB/D-His for GG RecD_His_SapI_rv & RecB_His_SapI_rv expect: 5,3 kb repeated because didnt work RecC-His for GG RecB-RecC-UTR_SapI_rv & RecC_His_SapI_fw expect: 3,5 kb repeated because didnt work TM: all 60 ° |
| Results: | 1,2,4 worked 3,5,6 didnt PIC |
redo: Assembling pSB1C3_RecBCD(WT) with Gibson Assembly
2018/08/08| Participants: | Enikö Baligács, Katja Neishsalo |
| Protocol: | PCR, Agarose gel, Gel purification, Gibson Assembly, Chemical transformation |
| Notes: |
BB & LacO
Primer: GA_lacO_rv & GA_lact_fw
expect: 2,5 kb RecC-UTR for GA GA_RecC_fw & GA_RecB_rv expect: 3,5 kb RecB/D for GA GA_RecB_fw & GA_RecD_rv expect: 5,3 kb; repeated because didnt work BB & LacO for GG SapI Lact_His_SapI_fw & RecC_lacO_His_SapI_rv expect: 2,5 kb RecB/D-His for GG RecD_His_SapI_rv & RecB_His_SapI_rv expect: 5,3 kb repeated because didnt work RecC-His for GG RecB-RecC-UTR_SapI_rv & RecC_His_SapI_fw expect: 3,5 kb repeated because didnt work TM: all 60 ° |
| Results: | 1,2,4,6 worked (PIC) |
DNA mini-preparation of pSB1C3_mRFP
2018/08/09| Participants: | Enikö Baligács |
| Protocol: | Miniprep |
testing pSB1C3_RecBCD(WT) toxicity in cells
2018/08/13| Participants: | Katja Neishsalo |
| Protocol: | Chemical transformation, Gibson Assembly, Agarose gel |
| Notes: | To improve cell survival we added 1% glucose to lb media |
| Results: | No colonies? |
Genomic extraction of RecBC fragment
2018/08/14| Participants: | Katja Neishsalo |
| Protocol: | Restriction digest |
| Notes: | Primers |
| Results: | Gel PIC |
redo: Assembling pSB1C3_RecBCD(WT) with Gibson Assembly
2018/08/14| Participants: | Enikö Baligács, Katja Neishsalo |
| Protocol: | PCR, Agarose gel, Gel purification, Gibson Assembly, Chemical transformation |
| Notes: |
BB & LacO
Primer: GA_lacO_rv & GA_lact_fw
expect: 2,5 kb RecC-UTR for GA GA_RecC_fw & GA_RecB_rv expect: 3,5 kb RecB/D for GA GA_RecB_fw & GA_RecD_rv expect: 5,3 kb; repeated because didnt work BB & LacO for GG SapI Lact_His_SapI_fw & RecC_lacO_His_SapI_rv expect: 2,5 kb RecB/D-His for GG RecD_His_SapI_rv & RecB_His_SapI_rv expect: 5,3 kb repeated because didnt work RecC-His for GG RecB-RecC-UTR_SapI_rv & RecC_His_SapI_fw expect: 3,5 kb repeated because didnt work TM: all 60 ° |
| Results: | We lost the samples during agarose gel verification because the loading dye was contaminated. Redoing the experiment with Thomas (supervisor) yielded no colonies. Eni decided to go to a different lab because cloning doesnt work at the simmel lab. We therefore went to Prof. Gil Westmeyer at the Helmholtz Zentrum and continued our work there. |
preparing different pSB backbones
2018/08/20| Participants: | Julia Mayer, Thomas Frank |
| Protocol: | PCR, Agarose gel, Gel extraction |
| Notes: | |
| Results: | no results |
Assembling pSB1C3_RecBCD(WT) with Gibson Assembly
2018/08/27| Participants: | Enikö Baligács |
| Protocol: | PCR, Agarose gel, Gel purification, Gibson Assembly, Chemical transformation |
| Notes: | RecBD: RecD_extract_fw & RecB_extract_rv Template: genomic DNA (54,5 ng/µl) RecC: RecC_extract_fw & RecC_extract_rv Template: genomic DNA (54,5 ng/µl) BB & LacO Primer: GA_lacO_rv & GA_lact_fw expect: 2,5 kb RecC-UTR for GA GA_RecC_fw & GA_RecB_rv expect: 3,5 kb RecB/D for GA GA_RecB_fw & GA_RecD_rv expect: 5,3 kb; repeated because didnt work BB & LacO for GG SapI Lact_His_SapI_fw & RecC_lacO_His_SapI_rv expect: 2,5 kb RecB/D-His for GG RecD_His_SapI_rv & RecB_His_SapI_rv expect: 5,3 kb repeated because didnt work RecC-His for GG RecB-RecC-UTR_SapI_rv & RecC_His_SapI_fw expect: 3,5 kb repeated because didnt work TM: all 60 ° |
| Results: | Colonies for pSB1C3_RecBCD(WT), no colonies for pSB1C3_RecBCD-His |
Redo: Assembling pSB1C3_RecBCD-His
2018/08/29| Participants: | Enikö Baligács |
| Protocol: | PCR, Agarose gel, Gel purification, Gibson Assembly, Chemical transformation |
| Notes: | Primers??; We used T4 ligase instead of quick ligase like before. We added GC-buffer to the PCR reaction |
| Results: | No colonies for pSB1C3_RecBCD-His |
sequencing pSB1C3_RecBCD(WT)
2018/08/30| Participants: | Enikö Baligács |
| Protocol: | Miniprep, Sequencing, PCR |
| Notes: | |
| Results: | sequencing didnt give reads, but because eurofins had problems we prepared new DNA and told them to do it again. results?. sequencial test-PCR showed a correctly assembled plasmid of recBCD(WT) (PIC) |