Difference between revisions of "Team:Munich/recbcdwt.html"
Franziwinzig (Talk | contribs) |
Franziwinzig (Talk | contribs) |
||
| Line 23: | Line 23: | ||
<td>Notes:</td> | <td>Notes:</td> | ||
<td> | <td> | ||
| − | + | 1) PCR for Backbone with Lac-Operon and Terminator: GA_lacO_rv & GA_lact_fw expect: 2,5 kb <br>2) PCR of RecC for Gibson assembly with Primers: GA_RecC_fw & GA_RecB_rv expect: 3,5 kb ; <br>3) PCR of RecBD for Gibson Assembly with Primers: GA_RecB_fw & GA_RecD_rv expect: 5,3 kb; repeated because didnt work <br>4) PCR of Backbone with Lac-Operon and terminator for digestion and Ligation to RecBCD-His: Primers: Lact_His_SapI_fw & RecC_lacO_His_SapI_rv expect: 2,5 kb <br>5) PCR of RecBD for digestion and Ligation to RecBCD-His with Primers: RecD_His_SapI_rv & RecB_His_SapI_rv expect: 5,3 kb repeated because didnt work <br>6) PCR of RecC for digestion and ligation to RecBCD-His with Primers: RecB-RecC-UTR_SapI_rv & RecC_His_SapI_fw<br> expect: 3,5 kb repeated because didnt work; <br>TM for all: 60 °C; <br>PCRs 1 – 3 were used for a Gibson Ligation, PCRs 4-5 were digested with SapI and then Ligated. Both Ligation and Gibson product were transformed. on Gel: from left to right: PCR 1-6 | |
| − | + | ||
| − | expect: 2,5 kb <br> | + | |
| − | RecC | + | |
| − | GA_RecC_fw & GA_RecB_rv | + | |
| − | expect: 3,5 kb <br> | + | |
| − | + | ||
| − | GA_RecB_fw & GA_RecD_rv | + | |
| − | expect: 5,3 kb; repeated because didnt work <br> | + | |
| − | + | ||
| − | Lact_His_SapI_fw & RecC_lacO_His_SapI_rv | + | |
| − | expect: 2,5 kb <br> | + | |
| − | + | ||
| − | RecD_His_SapI_rv & RecB_His_SapI_rv | + | |
| − | expect: 5,3 kb | + | |
| − | RecC-His | + | |
| − | RecB-RecC-UTR_SapI_rv & RecC_His_SapI_fw | + | |
| − | expect: 3,5 kb | + | |
| − | TM | + | |
</td> | </td> | ||
</tr> | </tr> | ||
| Line 50: | Line 32: | ||
<img src="https://static.igem.org/mediawiki/2018/6/6c/T--munich--20180806_PCR1-6.jpg | <img src="https://static.igem.org/mediawiki/2018/6/6c/T--munich--20180806_PCR1-6.jpg | ||
" class="figure-img img-fluid rounded" alt=" "> | " class="figure-img img-fluid rounded" alt=" "> | ||
| − | <figcaption class="figure-caption"></figcaption> | + | <figcaption class="figure-caption">on Gel: from left to right: PCR 1-6</figcaption> |
</figure> | </figure> | ||
</td> | </td> | ||
Revision as of 20:35, 17 October 2018
Cloning of RecBCD-WT and RecBCD-His-Tag
2018/08/06 – 2018/08/10| Participants: | Enikö Baligács, Katja Neishsalo |
| Protocol: | PCR, Agarose gel, Gel purification, Gibson Assembly, Chemical transformation |
| Notes: |
1) PCR for Backbone with Lac-Operon and Terminator: GA_lacO_rv & GA_lact_fw expect: 2,5 kb 2) PCR of RecC for Gibson assembly with Primers: GA_RecC_fw & GA_RecB_rv expect: 3,5 kb ; 3) PCR of RecBD for Gibson Assembly with Primers: GA_RecB_fw & GA_RecD_rv expect: 5,3 kb; repeated because didnt work 4) PCR of Backbone with Lac-Operon and terminator for digestion and Ligation to RecBCD-His: Primers: Lact_His_SapI_fw & RecC_lacO_His_SapI_rv expect: 2,5 kb 5) PCR of RecBD for digestion and Ligation to RecBCD-His with Primers: RecD_His_SapI_rv & RecB_His_SapI_rv expect: 5,3 kb repeated because didnt work 6) PCR of RecC for digestion and ligation to RecBCD-His with Primers: RecB-RecC-UTR_SapI_rv & RecC_His_SapI_fw expect: 3,5 kb repeated because didnt work; TM for all: 60 °C; PCRs 1 – 3 were used for a Gibson Ligation, PCRs 4-5 were digested with SapI and then Ligated. Both Ligation and Gibson product were transformed. on Gel: from left to right: PCR 1-6 |
| Results: | 1,2,4 worked
3,5,6 didnt 
|
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, Ligation |
| 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: | Obtained more RecBD extracted from the Genome. |
redo: Assembling pSB1C3_RecBCD(WT) with Gibson Assembly
2018/08/14| Participants: | Enikö Baligács, Katja Neishsalo |
| Protocol: | Ligation, Chemical transformation |
| Notes: | BB & LacO The fragments obtained before (PCR 4-6) were Ligated. This time T4 ligase was used instead of Quick Ligase. |
| 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 |
| 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 – 2018/09/11| Participants: | Enikö Baligács |
| Protocol: | Miniprep, Sequencing, PCR |
| Notes: | Two different test PCRs: Primer pair 1): Seq_RecC8_rv and VR -> expected 7 kb 2) Seq_RecB_fw_6 and VF2 -> expected 4 kb ; both with longAmp polymerase; AT: 58 °C; ET: 6 minutes. |
| 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) |