Difference between revisions of "Team:Munich/recbcdwt.html"
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<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>Cloning work was continued at Prof. Gil Westmeyer's Lab at the Helmholtz Zentrum due to potential problems with contamination of samples and problems with competent cells.</td> |
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</table> | </table> | ||
Latest revision as of 03:02, 18 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 Miniprep 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: | Cloning work was continued at Prof. Gil Westmeyer's Lab at the Helmholtz Zentrum due to potential problems with contamination of samples and problems with competent cells. |
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 |
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) |