Team:Stuttgart/Notebook
June
29.06.
Present: Stefanie, Lina
Lab Work:
1 T75 bottle with HeLa received from the Institute of Cell Biology and Immunology.
30.06.
Present: Stefanie, Lina
Lab Work:
Transferred to our incubator (37°C and 5% CO2).
Medium accepted.
3 ml trypsin and EDTA, incubated for 7 minutes in the incubator.
Supernatant distributed on tubes and centrifuged.
Pellet dissolved in medium.
In T175 bottle with 17 ml medium.
July
01.07.
Present: Sumohit, Ida, Miguel, Ben
Lab Work:
Production of agar medium and LB medium.
02.07.
Present: Stefanie, Lina
Lab Work:
HeLa cells split from 1 T175 to 3 T175.
like 30.06.
07.07.
Present: Stefanie, Lina
Lab Work:
HeLa cells split from 1 T175 to 3 T175.
like 30.06.
09.07.
Present: Stefanie, Lina
Lab Work:
Contamination in HeLa incubator.
13.07.
Present: Stefanie, Luisa
Lab work:
HeLa cells splitting.
14.07.
Present: Sumohit, Ida, Miguel, Ben
Lab Work:
Processing of the ordered primers and fragments for the E. coli fragments and primers.
PCR with wildtype dsup fragment (F1), C-terminal dsup fragment (F2), N-terminal dsup fragment (F3) and pBAD18.
15.07.
Present: Sumohit, Ida, Ben, Stefanie, Lina
Lab Work:
Preparation of agarose.
Gel electrophoresis at 100 V, 75mA and 45 minutes.
Cleaning of incubator.
New HeLa cells fetched.
Preparation of trypsin.
16.07.
Present: Sumohit, Ida, Lina, Luisa
Lab Work:
DNA gel extraction from the agarose gel from 15.07.
Gibson assembly.
DNA clean-up and concentration.
HeLa cells splitting.
19.07.
Present: Sumohit, Ben, Miguel
Lab Work:
Transformation into DH5α competent cells.
Preparation of Ampicillin solution.
20.07.
Present: Stefanie, Lina
Lab work:
HeLa cells splitting.
21.07.
Present: Sumohit, Ida, Miguel
Lab Work:
No colonies on the agar plates.
PCR with F1, F2, F3 and pBAD18.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
22.07.
Present: Sumohit, Ida
Lab Work:
DNA gel extraction from the agarose gel from 21.07.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
pBAD18 plasmid isolation.
23.07.
Present: Sumohit, Miguel, Ben, Lina
Lab work:
No colonies on the agar plates.
HeLa cells splitting.
27.07.
Present: Sumohit, Miguel, Stefanie, Ben
Lab Work:
HeLa cells splitting.
For HeLa Gibson:
Processing of the ordered primers and fragments for the HeLa fragments and primers.
For E. coli Gibson:
No colonies on the agar plates.
PCR with F1, F2, F3, pBAD18, fragment for HeLa (FHL) and pcDNA3.1.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
28.07.
Present: Sumohit, Miguel, Ben
Lab Work:
For HeLa Gibson:
PCR with fragment for HeLa (FHL) and pcDNA3.1.
For E. coli Gibson:
DNA gel extraction from the agarose gel from 27.07.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
29.07.
Present: Sumohit, Miguel
Lab Work:
For E. coli Gibson:
No colonies on the agar plates.
30.07.
Present: Stefanie, Lilli, Sumohit, Miguel
Lab Work:
HeLa cells splitting.
For HeLa Gibson:
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
DNA gel extraction from the agarose gel.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
August
03.08.
Present: Ben, Lilli
Lab work:
HeLa cells splitting.
04.08. and 05.08.
Present: Sumohit, Ben
Lab Work:
For E. coli Gibson:
PCR with F1, F2, F3 and pBAD18.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
DNA gel extraction from the agarose gel.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
05.08.
Present: Stefanie, Lilli
Lab work:
Several bottles split, bem rest medium changed.
Seeding: 2 ml each with 20,000-50,000 cells.
Counting chamber: count corner chambers --> mean value
06.08.
Present: Sumohit, Lilli
Lab Work:
HeLa cells splitting.
For HeLa Gibson:Plasmid isolation.
Nanodrop --> Concentration is too low for sequencing.
DNA clean-up and concentration for better concentration.
ent for sequencing.
Sample names:
P1F --> Plasmid pcDNA3.1 1st with forward Primer
P1R --> Plasmid pcDNA3.1 1st with reverse Primer
P2F --> Plasmid pcDNA3.1 2nd with forward Primer
P2R --> Plasmid pcDNA3.1 2nd with reverse Primer
14:00 to Mr Eisler --> 1 sample was set under microscope- setting of 2 blocks: 1st block normal light 48 hours; 2nd Blocj UV 7 sec all for 24 hours minimal.
09.08.
Present: Sumohit, Ben
Lab work:
Plasmid irradiation test.
Colony PCR.
10.08.
Present: Sumohit, Ben, Stefanie
Lab work:
HeLa cells splitting.
Plasmid isolation.
Plasmid irradiation test.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
Colony PCR.
11.08.
Present: Sumohit, Miguel
Lab work:
Production of agar medium and LB medium.
PCR for plasmid isolation test.
12.08 and 13.08.
Present: Sumohit, Miguel, Lilli
Lab Work:
HeLa cells splitting.
For E. coli and HeLa Gibson (From now with NEBuilder):
PCR with F1, F2, F3, FHL, pcDNA3.1 and pBAD18.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
DNA gel extraction from the agarose gel.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
16.08 and 17.08.
Present: Sumohit, Ben, Stefanie, Lina
Lab Work:
HeLa cells splitting.
Colony-PCR with colonies on the agar plates from 12.08 and 13.08
Begin of new live-cell imaging.
For E. coli and HeLa Gibson:
PCR with F1, F2, F3, FHL, pcDNA3.1 and pBAD18.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
DNA gel extraction from the agarose gel.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
18.08. and 19.08.
Present: Stefanie, Lina
Lab Work:
HeLa cells splitting.
For E. coli and HeLa Gibson:
PCR with F1, F2, F3, FHL, pcDNA3.1 and pBAD18.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
DNA gel extraction from the agarose gel.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
Begin of new live-cell imaging.
20.08.
Present: Sumohit, Ida
Lab work:
Production of competent cells.
Transformation into DH5α competent cells.
Plasmid irradiation experiment.
23.08.
Present: Stefanie, Lina, Lilli
Lab work:
Colony-PCR with colonies on agar plate form 18. and 19.08.
Production of competent cells.
Preparation of Ampicillin solution.
24.08.
Present: Stefanie, Lina, Lilli
Lab work:
HeLa cells splitting.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
27.08.
Present: Stefanie, Lina, Lilli
Lab work:
HeLa cells splitting.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
31.08.
Present: Sumohit, Miguel, Lina
Lab work:
HeLa cells splitting.
Plasmid irradiation experiment.
PCR with irradiated fragments.
September
02.09.
Present: Stefanie, Lina
Lab work:
HeLa life cell imaging.
06.09.
Present: Sumohit, Lina
Lab work:
Production of agar medium and LB medium.
07.09.
Present: Sumohit, Ben
Lab work:
HeLa cells splitting.
Plasmid isolation.
For E. coli Gibson:
PCR with F1, F2, F3 and pBAD18.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
DNA gel extraction from the agarose gel.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
08.09.
Present: Sumohit, Ida, Ben
Lab work:
Plasmid irradiation experiment.
PCR with irradiated fragments.
Production of agar medium.
Plasmid isolation from overnight culture with single colonies from the agar plates of 07.09.
Measurement of DNA concentration with Nanodrop.
Sent for sequencing.
Sample names:
Dsup WT
Dsup C-Term
Dsup N-Term
10.09.
Present: Ben, Stefanie, Sumohit
Lab work:
HeLa cells splitting.
For HeLa Gibson:
PCR with FHL and pcDNA3.1.
Preparation of agarose.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
For E. coli:
Transformation into BW25113 competent cells.
Production of TB salts and TB medium.
13.09.
Present: Ben, Stefanie, Sumohit
Lab work:
HeLa cells splitting.
DNA gel extraction from the agarose gel.
Gibson assembly.
DNA clean-up and concentration.
Transformation into DH5α competent cells.
For E. coli:
Preparation for the expression of wildtype dsup and N-terminal dsup.
14.09.-15.09.
Present: Sumohit, Ben
Lab work:
Expression of wildtype dsup and N-terminal dsup.
16.09.
Present: Sumohit, Lina
Lab work:
Harvest cells and store in saline until cell breaking.
SDS-PAGE.
14.09.
Present: Lilli, Lina
Lab work:
HeLa cells splitting.
17.09.
Present: Lina, Sumohit
Lab work:
HeLa cells splitting.
Preparation for the expression of genotypes for E. coli.
SDS-PAGE.
Plasmid isolation for HeLa Gibson.
20.09.-24.09.
Present: Sumohit, Lina
Lab work:
Expression of genotypes for E. coli.
Harvest cells and store in saline until cell breaking.
On 24.09. SDS-PAGE.
21.09.
Present: Ben, Stefanie
Lab work:
HeLa cells splitting.
24.09.
Present: Lilli, Ben
Lab work:
HeLa cells splitting.
26.09.
Present: Sumohit, Lina
Lab work:
Harvest cells and store in saline until cell breaking.
SDS-PAGE.
28.09.
Present: Lina, Lilli, Sumohit
Lab work:
HeLa cells splitting.
Preliminary tests for intracellular plasmid irradiation.
PCR with FHL and pcDNA3.1.
Preparation of agarose gel.
Gel electrophoresis at 100 V, 75 mA and 45 minutes.
30.09.
Present: Sumohit
Lab work:
Cell disruption with high pressure homogeniser.
October
01.10.
Present: Lilli, Stefanie
Lab work:
HeLa cells splitting.
04.10.
Present: Lilli, Sumohit
Lab work:
HeLa cells splitting.
Protein purification in Äkta.
Production of agar medium.
Preliminary tests for intracellular-located dsup cells irradiation.
05.10.
Present: Lilli, Sumohit
Lab work:
Expression of N-terminal dsup by 30°C.
Preliminary tests for intracellular-located dsup cells irradiation.
06.10.
Present: Lilli, Sumohit
Lab work:
Harvest cells and cell disruption with high pressure homogeniser.
08.10.
Present: Lilli, Sumohit
Lab work:
HeLa cells splitting.
Protein purification in Äkta.
Experiment for intracellular-located dsup cells irradiation.
SDS-PAGE.
11.10.
Present: Lilli, Ben
Lab work:
OD measurement and SDS-PAGE of all expression samples.
12.10.
Present: Sumohit, Ida
Lab work:
HeLa cells splitting.
SDS-PAGE.
Experiment for intracellular-located dsup cells irradiation.
13.10.
Present: Lilli, Luisa, Marcel
Lab work:
Bradford assay.
Experiment for intracellular-located dsup cells irradiation.
Preparation of LB medium.
14.10.
Present: Lilli, Ida, Ben
Lab work:
SDS-PAGE
Preparation for experiment for extracellular-located dsup irradiation.
15.10.
Present: Lilli, Luisa, Ida
Lab work:
SDS-PAGE.
Experiment with extracellular-located dsup irradiation.
Experiment with intracellular-located dsup irradiation.
18.10.
Present: Lilli, Stefanie
Lab work:
Sterile filtered sample C-term peak 2
Bradford assay.
HeLa cells splitting.
Experiment for extracellular-located dsup irradiation.
Preparing overnight culture.
19.10.
Present: Lilli, Lina
Lab work:
Experiment for intracellular-located dsup irradiation
SDS-PAGE.
20.10.
Present: Lina, Lilli
Lab work:
Experiment for extracellular-located dsup irradiation.