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
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Read-out
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Wednesday 22nd
Cas13a123
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Cas13a41234
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Thursday 23rd
REad-out
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Friday 24th
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Monday 27th
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Tuesday 28th
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Wednesday 29th
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Thursday 30th
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Friday 31rd
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Thursday 1st
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Friday 2nd
Total RNA extraction of E. coli W3110 with Norgen kit
Protocol: Total RNA Purification with Norgen Kit
Participants: Jorge
Observations:
Instead of using TE and lysozyme to lyse the cells, they were resuspenden in 2% SDS in TAE and incubated at 75 ºC for 15 min.
The 6 samples were stored at -80 ºC and labeled T RNA #1-6
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Monday 5th
Target
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RNA Adsorption Test on first 3D print
Participants: Jorge
Notes:
Total RNA sample 5 used, prepared on Friday 02/06 by Jorge (Concentration: 126 ng/µl)
Procedure:
Take 1 µl presample from stock ; add with 2 µl nf H2O and 3 µl 2x RNA LD
Put 5 µl of sample on device
Take 1 µl of sample immediately, add with 2 µl nf H2O and 3 µl 2x RNA LD
Wait 15 minutes
Take 1 µl of sample immediately, add with 2 µl nf H2O and 3 µl 2x RNA LD
Store at -80 °C
What still needs to be done:
Cook samples at 95 °C for 10 minutes and put directly on ice afterwards (to prevent refolding)
Load on Gel and quantify
Tuesday 6th
Target
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Wednesday 7th
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Thursday 8th
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Friday 9th
Target
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Monday 12th
Target
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Tuesday 13th
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Lysis-tests with acidic phenol chlorophorm extraction
Protocol: Alkaline lysis
Protocol: Phenol-Chlorophorm extraction
Protocol: Trizol Reagenz protocol
Participants: Julian, Patrick
Notes:
Three lysis methods were used: Alkaline Lysis, heat lysis with SDS and Trizol-Lyse.
No vortexing for lysis (may break up cells and won't be available in our device), only room-temperature (apart form SDS as reference)
Cells had OD of 5.97 -> 4.78*10^9 cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158
Cells had OD of 5.97 -> 4.78*10^9 cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158
-> dilluted 1:100 (to V=10mL) and pelleted 209 miL to get to 10^7 cells
A
B
C
D
1
Method SDS NaOH TRIzol 2
prepare 1 mL TES (10mM Tris-HCl, 10mM EDTA, pH 8.0, 2% SDS -> TE-Buffer + SDS... 3
----Lysis 1x 3x 1x 4
separate to tubes Spin 300xG, 5 min ->Ice, TROzol to -20! (100 miL at OD 1 -> 10^7 cells, Max. according to TRIzolPaper 100 miL bactSusp 100 miL bact-susp 100 miL bact-suspension 5
No washing step, bad for mRNA (TRIzolP.) 300 µl (0.45 ml) TES resuspend pellet 6
12,5 uL Inhibitor (40k U/ml, 1 U/miL required) 15 min, 75 °C lysis, vortex one probe after another 7
(Addition of inhibitor); Transform total volume to gel tube resuspend thorougly in 66 µl (100 miL) Solution1 8
132 µl (200 miL) Sol2, INVERT 4x 9
wait 0, 1 ,3 min 10
add 99 µl (150 miL) Sol3, INVERT 4x 11
Total volume: 297 µl (0.45 ml) 12
--phenChloExtr 13
measure pH, add acid... meassure Trizol PH, add acid if needed (pH <5) 14
add 600 µl [same V (0.45 ml)] Phe:chloro. add 1 mL TRIzol to 10^7 cells 15
5 min incubation 16
add 0.2 mL CHLOROPHORM 17
incub 2.5 (2-3) min, RT 18
centri 12k g 15 min 4°C 19
angle 45°, extract top phase carefully -> discard everything else, prot & DNA -> which Vol for the TRIzol aqueous phase? 20
add 0.4 (0.375) ml Isopropanol, wait 10 min, RT 21
centrifuge 10 min 12k , 4°C -> 22
Aditional step, because RNA pellet was not entierly spinned down: Centrifugation (16000 rcf, 5 min, 4 °C) big-pellet assay in fridge, but 1. interphase was punctured/gel(DNA?) stuck to pipette 2. s.u. 23
Aditional step, because suspected RNA fog was not entirely spinned down: remove upper and lower RNA fog phase 24
Additional step: Centrifugation (16000 rcf, 5 min, 4 °C) 25
No RNA pellet or RNA fog any more :( 26
Addition of 400 µl isopropanol -> No RNA pellet or fog any more ->prob. removed too much 27
discard supernatant -> RNA in gel-like pellet! -> gel hardly visible, nearly draged it out of tube (stuck to pipette...) 28
resuspend in 0.75 ml 70 % (75% ideally) ethanol -> storeed RNA at -20°C is stable up to 1 year... 29
vortex, centri 5 min 7,5k 4° 30
discard supernatant, air dry for 5-10 min (do NOT let completely dry out) 31
resuspend in RNAse free water 30 µl (20-50 miL) ->measurement...
Results
No pellets seen during procedure, concentration too low for photometer -> repeat with more cells
Wednesday 14th
Target
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Thursday 15th
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Friday 16th
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Lysis-tests with acidic phenol chlorophorm extraction
Protocol: Lysis test pipetting scheme
Protocol: Phenol-Chlorophorm extraction
Participants: Julian, Patrick
Notes:
Cell suspension had OD600 of 2,220 -> 1.78 x 10^9 cells/ml according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158
Experiments were done with 108 and 109 cells per sample, because it failed with 10^7 cells last time
Aspiration of supernatant in phenol-chlorophorm extraction: about 2/3 aspirated (200 µl) and 1/3 (100 µl) left to not destroy interphase
No pellet after RNA precipitation (-80 °C) and subsequent centrifugation in:
NaOH, 1 min, 10^8
SDS, 10^9
Trizol, 10^8
Mistakes:
SDS, 10^9: aspirated 100 µl instead of 200 µl supernatant
NaOH, 10^8, 3 min: contamination with DNA (<10% DNA interphase transfered)
NaOH, 10^9, 3 min: added + 200 µl of SDS 109 tube
Results
Sample
[] [µg/ml]
adjusted C**
A260/A280
A260/A230
A230 [A]
A260 [A]
A280 [A]
A320 [A]
1
SDS, 10^8 cells 36.8 55 1.704 2.044 0.047 0.094 0.056 0.002 2
SDS, 10^9 cells* 119 357 1.693 2.069 0.146 0.300 0.178 0.002 3
NaOH, 0 min, 10^8 cells 35.2 53 1.725 2.146 0.042 0.089 0.052 0.001 4
NaOH, 0 min, 10^9 cells 586 879 1.923 1.993 0.740 1.470 0.767 0.005 5
NaOH, 1 min, 10^8 cells 73.6 110 1.235 0.586 0.317 0.187 0.152 0.003 6
NaOH, 1 min, 10^9 cells 370 555 1.945 1.958 0.475 0.927 0.478 0.003 7
NaOH, 3 min, 10^8 cells* 259 389 1.849 1.491 0.439 0.652 0.355 0.005 8
NaOH, 3 min, 10^9 cells* 275 413 1.850 2.212 0.316 0.693 0.368 0.005 9
TRIzol, 10^8 cells 194 265 1.362 0.221 2.200 0.487 0.358 0.002 10
TRIzol, 10^9 cells 160 218 1.429 0.488 0.822 0.403 0.283 0.003
Measured [RNA] by Implen Nanophotometer (20170616).
**adjusted for different supernatant V aspirated at extraction step
Monday 19th
Target
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Tuesday 20th
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Wednesday 21st
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Thursday 22nd
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Friday 23rd
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Monday 26th
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Tuesday 27th
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Wednesday 28th
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Thursday 29th
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Friday 30th
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Tuesday 1st
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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.
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Thursday 3rd
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Friday 4th
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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
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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.
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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.
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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
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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
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Monday 14th
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Tuesday 15th
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Wednesday 16th
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Thursday 17th
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Friday 18th
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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:
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Monday 28th
Target
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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)
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Thursday 31st
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