Team:Munich/recbcdwt.html

Assembling pSB1C3_RecBCD(WT) with gibson assembly

2018/08/06

Participants:	eni, katja
Protocol:	PCR,Agarose Gel, Gel purification, Gibson assembly, chem trafo
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:	eni, katja
Protocol:	PCR,Agarose Gel, Gel purification, Gibson assembly, chem trafo
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:	Eni
Protocol:	mini-prep

testing pSB1C3_RecBCD(WT) toxicity in cells

2018/08/13

Participants:	Katja
Protocol:	chem trafo, 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
Protocol:	PCR
Notes:	Primers
Results:	Gel PIC

redo: Assembling pSB1C3_RecBCD(WT) with gibson assembly

2018/08/14

Participants:	Enikö, Katja
Protocol:	PCR,Agarose Gel, Gel purification, Gibson assembly, chem trafo
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, Thomas
Protocol:	PCR, Agarose gel, gelextraction
Notes:
Results:	?

Assembling pSB1C3_RecBCD(WT) with gibson assembly

2018/08/27

Participants:	Eni
Protocol:	PCR, Agarose Gel, Gel purification, Gibson assembly, chem trafo
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
Protocol:	PCR, Agarose Gel, Gel purification, Gibson assembly, chem trafo
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
Protocol:	mini-prep, 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)