Team:Munich/recbcdwt.html
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: |
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 |
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) |