Difference between revisions of "Team:Munich/recbcdwt.html"

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     <figcaption class="figure-caption">on Gel: from left to right: PCR 1-6</figcaption>
 
     <figcaption class="figure-caption">on Gel: from left to right: PCR 1-6</figcaption>
 
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    <figcaption class="figure-caption">Repeated PCRs 3, 5 and 6:</figcaption>
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Revision as of 20:36, 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
on Gel: from left to right: PCR 1-6
Repeated PCRs 3, 5 and 6:

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)