Labbook
According to all known laws
of aviation,
there is no way a bee
should be able to fly.
Its wings are too small to get
its fat little body off the ground.
The bee, of course, flies anyway
because bees don't care
what humans think is impossible.
Yellow, black. Yellow, black.
Yellow, black. Yellow, black.
Cas13a
Read-out
Target
Other
Tuesday 21st
Cas13a
Protocol: protocol
Participants:
Observations:
Read-out
Protocol: Protocol
Participants:
Observations
Results
Wednesday 22nd
Cas13a123
Protocol: protocol
Participants:
Observations:
Cas13a41234
Protocol: Protocol
Participants:
Observations
Results
Thursday 23rd
REad-out
Protocol: protocol
Participants:
Observations:
Target
Protocol: Protocol
Participants:
Observations
Results
Friday 24th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Monday 27th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Tuesday 28th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Wednesday 29th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Thursday 30th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Friday 31rd
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Tuesday 1st
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Wednesday 2nd
InterLab Study calibration
Protocol: IGEM HQ InterLab Study Protocol
Participants: Dawafuti Erika
Observations:
Patch length correction was on in the plate reader.
Calibration for the plate reader experiment done with LUDOX and Fluorescein.
We saw no difference between LUDOX and water when measuring at an OD of 600 nm.
For the fluorescein measurement, the best gain was 600 using 485 nm/520 nm.
Plasmids from the plate 7 (positive and negative control, and devices 1-6) were resuspended.
readout
Protocol: Protocol
Participants:
Observations
Results
Thursday 3rd
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Friday 4th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Monday 7th
InterLab Study Day 1: Transformation
Protocol: IGEM HQ InterLab Study Protocol
Participants: Dawafuti Erika
Observations:
Each plasmids was transformed two times: with chemical transformation and with electroporation, for a total of 16 plasmids.
Results
Every plate except the ones for Device 1 had colonies.
readout
Protocol: Protocol
Participants:
Observations
Results
Tuesday 8th
InterLab Study Day 2: Colony transfer to liquid culture
Protocol: IGEM HQ InterLab Study Protocol
Participants: Dawafuti Erika
Observations:
Device 1 was again transformed, as no colonies grew on the plate.
Liquid cultures for the 7 plasmids whose plates showed colonies (all except Device 1) were made in duplicate.
The plates were stored at -4 °C.
readout
Protocol: Protocol
Participants:
Observations
Results
Wednesday 9th
InterLab Study Day 3: Plate reader experiments
Protocol: IGEM HQ InterLab Study Protocol
Participants: Dawafuti Erika
Observations:
After measuring the OD600 of the O/N cultures, they were diluted with LB+Cm to an OD600 of 0.02, in duplicates.
The absorbance and fluorescence of the diluted cultures were measured at 0, 2, 4 and 6 hours after the cultures were set.
Patch length correction was on in the plate reader.
Device 1 was again transformed, as no colonies grew on the plate.
Results
Transformation of Device 1 was successful.
readout
Protocol: Protocol
Participants:
Observations
Results
Thursday 10th
InterLab Study Day 4: Preparation of O/N cultures for plate reader experiments
Protocol: IGEM HQ InterLab Study Protocol
Participants: Dawafuti Erika
Observations:
O/N liquid cultures in duplicate were set up from all 8 Devices.
readout
Protocol: Protocol
Participants:
Observations
Results
Friday 11th
InterLab Study Day 5: Plate reader calibration and experiments
Protocol: IGEM HQ InterLab Study Protocol
Participants: Dawafuti Erika
Observations:
Patch length correction was off in the plate reader.
Calibration for the plate reader experiment done with LUDOX and Fluorescein.
We saw the difference in measurements between LUDOX and water.
After measuring the OD600 of the O/N cultures, they were diluted with LB+Cm to an OD600 of 0.02, in duplicates.
The absorbance and fluorescence of the diluted cultures were measured at 0, 2, 4 and 6 hours after the cultures were set.
Results
readout
Protocol: Protocol
Participants:
Observations
Results
Monday 14th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Tuesday 15th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Wednesday 16th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Thursday 17th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Friday 18th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Monday 21st
FINA extraction for DNA with E. coli W3110 pre-purified gDNA
Protocol: FINA Extraction for DNA
Participants: Jorge
Observations:
The gDNA was pre-purified with the Phenol/Chloroform method.
The gDNA solution was diluted 1:100, 1:1000, 1:100000, 1:10e6 and 1:10e8. Each dilution was then used as a sample for the FINA extraction.
From each dilution, two extractions were performed. One as in protocol (1N-5N), the other without the NaOH washing step (1-5).
As negative control (KK) FINA extraction was performed as in protocol with nuclease free water as sample.
Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples and purified gDNA from 21.08.17
Protocol: PCR amplification with Q5 Master Mix
Participants: Jorge
Observations
For the membranes, no template was directly pipette. Instead, the membranes were put in the tube.
p-bGal_N_N was used as forward primer
p-bGal_N_C was used as reverse primer
Annealing was performed at 61 ºC
The amplification step was 90 s long
A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products
Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder
Results
1N-5N: 1:100-1:10e8 dilutions FINA extraction performed as in protocol.
1-5: 1:100-1:10e8 dilutions FINA extraction without washing step.
1K-5K: 1:100-1:10e8 dilutions.
KK: negative control for FINA extraction (nuclease free water as sample).
Tuesday 22nd
FINA extraction for DNA with E. coli W3110 cell culture
Protocol: FINA Extraction for DNA
Participants: Jorge
Observations:
The extraction was done when the culture had an OD600 of 1.2.
The cells were diluted 1:1, 1:100 and 1:1000 before the extraction.
Each dilution was used as sample twice: as in protocol (1N-3N and P1N-P3N) and washed with 300 ul lysis buffer from RNA extraction kit (Norgen, P/N 17200) (1G-3G and P1G-P3G).
Membranes 1N-3N and 1G-3G were put in 40 ul nuclease free water and incubated at RT for 10 min in order for the bounded DNA to go into solution.
Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 22.08.17
Protocol: PCR amplification with Q5 Master Mix
Participants: Jorge
Observations
The membranes from the FINA extraction were directly put into the PCR-mix.
No template DNA was directly pipetted.
p-bGal_N_N was used as forward primer.
p-bGal_N_C was used as reverse primer.
Annealing was performed at 61 ºC.
The amplification step was 90 s long.
A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder
Results
1N-3N: 1:1-1:1000 dilutions FINA extraction performed as in protocol, loaded in gel after elution (no PCR).
1G-3G: 1:1-1:1000 dilutions FINA extraction performed with lysis buffer as washing step, loaded in gel after elution (no PCR).
P1N-P3N: 1:1-1:1000 dilutions FINA extraction performed as in protocol.
P1G-P3G: 1:1-1:1000 dilutions FINA extraction with lysis buffer as washing buffer.
KK: negative control for FINA extraction (nuclease free water as sample).
Wednesday 23rd
FINA extraction for DNA with E. coli W3110 cell culture
Protocol: FINA Extraction for DNA
Participants: Jorge, Benedikt
Observations:
The extraction was done when the culture had an OD600 of 2.33.
The cells were diluted 1:1, 1:100, 1:10000, 1:10e6 and 1:10e8 before the extraction.
Each dilution was used as a sample for the FINA extraction. In addition, three 1:1 dilutions were used as samples for FINA extraction: two according to protocol and the third with 300 ul lysis buffer as washing buffer. One of the 1:1 membranes was put in nuclease free water and incubated at RT for 20 min in order for the bounded DNA to go into solution.
Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 23.08.17
Protocol: PCR amplification with Q5 Master Mix
Participants: Jorge, Benedikt
Observations
The membranes from the FINA extraction were directly put into the PCR-mix.
No template DNA was directly pipetted.
p-bGal_N_N was used as forward primer.
p-bGal_N_C was used as reverse primer.
Annealing was performed at 61 ºC.
The amplification step was 90 s long.
A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
Afte the PCR, only 2,5,L and KK were loaded in the gel, as the other tubes had no liquid.
The gel was loaded before it was submerged in TAE-buffer.
Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder
Results
2: 1:100 dilution FINA extraction performed as in protocol
L: 1:1 dilution FINA extraction performed with 300 ul lysis buffer as washing buffer.
KK: negative control for FINA extraction (LB-medium as sample).
Thursday 24th
FINA extraction for DNA with E. coli W3110 cell culture (Repetition of the experiment from 23.08.17)
Protocol: FINA extraction for DNA
Participants: Jorge, Benedikt
Observations:
As there were problems with the gel from the 23th, we repeated the experiment.
The extraction was done when the culture had an OD600 of 2.78.
The cells were diluted 1:1, 1:100, 1:10000, 1:10e6 and 1:10e8 before the extraction.
Each dilution was used as a sample for the FINA extraction. In addition, three 1:1 dilutions were used as samples for FINA extraction: two according to protocol and the third with 300 ul lysis buffer as washing buffer. One of the 1:1 membranes was put in nuclease free water and incubated at RT for 20 min in order for the bounded DNA to go into solution.
Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 24.08.17
Protocol: PCR amplification with Q5 Master Mix
Participants: Jorge, Benedikt
Observations
The membranes from the FINA extraction were directly put into the PCR-mix.
No template DNA was directly pipetted.
p-bGal_N_N was used as forward primer.
p-bGal_N_C was used as reverse primer.
Annealing was performed at 61 ºC.
The amplification step was 90 s long.
A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder
Results
1-5: 1:1-1:10e8 dilutions FINA extraction performed as in protocol
L: 1:1 dilution FINA extraction performed with 300 ul lysis buffer as washing buffer.
KK: negative control for FINA extraction (LB-medium as sample).
Total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with Norgen kit
Protocol: Total RNA Purification with Norgen Kit
Participants: Jorge, Benedikt
Observations:
2 tubes stored at -80 ºC. Labelled gRNA #1, gRNA #2.
Results
Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
Protocol: Phenol/Chloroform purification of RNA
Participants: Jorge
Observations:
The incubation step at -80 ºC was done, O/N.
2 tubes stored at -80 ºC. Labelled gRNA PEC #1, gRNA PEC #2.
Results
Friday 25th
Continuation of Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT from yesterday
Protocol: Phenol/Chloroform purification for RNA
Participants: Jorge
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Monday 28th
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
Tuesday 29th
FINA extraction for DNA with E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
Protocol: FINA extraction for DNA
Participants: Jorge
Observations:
The extraction was done when the culture had an OD600 of 0.88.
Na: Extraction as in protocol. Cl: Washed with 10 mM HCl instead of NaOH. H: incubated 10 min at 80 ºC. L: 10 min incubated in 1 mg/ml lysozyme. LH: 10 min incubated in 1 mg/ml lysozyme at 80 ºC.
Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 24.08.17
Protocol: PCR amplification with Q5 Master Mix
Participants: Jorge
Observations
The membranes from the FINA extraction were directly put into the PCR-mix.
No template DNA was directly pipetted.
p-bGal_N_N was used as forward primer.
p-bGal_N_C was used as reverse primer.
Annealing was performed at 61 ºC.
The amplification step was 90 s long.
A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder
Results
See Q5-PCR from 30.08.17
FINA extraction for RNA with purified total RNA (gRNA PEC #1)
Protocol: FINA extraction for RNA
Participants: Jorge
Observations:
First strand cDNA synthesis from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT purified total RNA (Phenol/Chloroform extraction from 24.08.17) and FINA-RNA eluate
Protocol: First strand cDNA synthesis
Participants: Jorge
Observations:
p-bGal_N_C was used as primer.
Sample gRNA PEC #1 from 24.08.17 was diluted 1:4 and used as sample P.
The eluate from the FINA extraction for RNA from 29.08.17 was used as sample S.
For both reactions (S and P), 6 ul sample were pipetted.
Wednesday 30th
Q5-PCR for beta-galactosidase from E. coli with cDNA samples from 29.08.17
Protocol: PCR amplification with Q5 Master Mix
Participants: Jorge
Observations:
p-bGal_N_N was used as forward primer.
p-bGal_N_C was used as reverse primer.
Annealing was performed at 61 ºC.
The amplification step was 90 s long.
A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder
Results
Na: FINA extraction performed as in protocol.
Cl: FINA extraction performed with 10 mM HCl instead of NaOH.
L: Cell solution incubated 10 min in 1 mg/ml lysozyme. No washing step in FINA
H: Cell solution incubated 10 min at 80 ºC. No washing step in FINA
LH: Cell solution incubated 10 min at 80 ºC in 1 mg/ml lysozyme. No washing step in FINA
K-: negative control FINA (LB-medium used as sample).
P: 1:4 dilution of gRNA PEC #1 after reverse transcription.
S: 1:4 dilution of gRNA PEC #1 after FINA extraction for RNA and reverse transcription.
KK: Negative control for the cDNA synthesis (water instead of MuLV-RT used)
readout
Protocol: Protocol
Participants:
Observations
Results
Thursday 31st
Target
Protocol: protocol
Participants:
Observations:
readout
Protocol: Protocol
Participants:
Observations
Results
