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
Wednesday 22nd
Thursday 23rd
Friday 24th
Monday 27th
Tuesday 28th
Wednesday 29th
Thursday 30th
Friday 31rd
Monday 3rd
Tuesday 4th
Wednesday 5th
Thursday 6th
Friday 7th
Monday 10th
Tuesday 11th
Wednesday 12th
Thursday 13th
Friday 14th
Monday 17th
Tuesday 18th
Wednesday 19th
Thursday 20th
Friday 21st
Monday 24th
Tuesday 25th
Plating of DH5α p2CT-His-MBP-Lbu-Cas13a-WT and DH5α p2CT-His-MBP-Lsh-Cas13a-WT on
LBCarb agar plates
Protocol: -
Participants: Ludwig
Observations:
Incubation at 37 °C over night.
Results
Bacteria grew and single colonies were visible.
Wednesday 26th
Preparation of 6 ml LBCarb cultures of DH5α p2CT-His-MBP-Lbu-Cas13a-WT and DH5α p2CT-His-MBP-Lsh-Cas13a-WT on LBCarb agar plates
Protocol: -
Participants: Ludwig
Observations:
Incubation at 37 °C over night.
Results
Bacteria cultures grew.
Thursday 27th
Preparation of cryo stocks for DH5α p2CT-His-MBP-Lbu-Cas13a-WT and
DH5α p2CT-His-MBP-Lsh-Cas13a-WT
Protocol: Cryo stocks
Participants: Ludwig
Minipreparation of DH5α Cas13a overnight cultures
Protocol: Minipreparation
Participants: Ludwig
Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT
in E. coli Rosetta.
Protocol: Electro transformation
Participants: Ludwig
Observations:
1 µl of plasmid DNA was used.
Bacteria were plated on LBCarb plates without additional Cm.
Results
Bacteria lost second plasmid probably.
Friday 28th
Monday 1st
Tuesday 2nd
Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT
in E. coli Rosetta.
Protocol: Electro transformation
Participants: Ludwig
Observations:
1 µl of plasmid DNA was used.
Bacteria were plated on LBCarb + Cm plates
Results
Bacteria grew and single colonies were visible.
Preparations of buffers for Cas13a purification.
Protocol: Protein purification
Participants: Chris, Ludwig, Julian
Observations:
TCEP changes the pH. Readjustement after adding neccessary.
Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT
in E. coli Rosetta.
Protocol: Electro transformation
Participants: Ludwig
Observations:
1 µl of plasmid DNA was used.
Bacteria were plated on LBCarb + Cm plates
Results
Bacteria grew, but only few single colonies were visible.
Wednesday 3rd
Electro transformation of p2CT-His-MBP-Lbu-Cas13a-WT and p2CT-His-MBP-Lsh-Cas13a-WT
in E. coli Rosetta.
Protocol: Electro transformation
Participants: Ludwig, Chris
Observations:
1 µl of plasmid DNA was used.
Bacteria were plated on LBCarb + Cm plates
Results
This time bacteria grew well and a lot of single colonies were visible.
Preparation of 25 ml LBCarb + Cm cultures of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT and Rosetta p2CT-His-MBP-Lsh-Cas13a-WT and incubated at 37 °C overnight.
Protocol: Protein Expression
Participants: Ludwig
Thursday 4th
Harvesting of Rosetta cells with overexpressed Cas13a Lbu and Cas13a Lsh
Protocol: Protein expression
Participants: Ludwig, Chris
Results
Bacterial pellets were stored at -80 °C.
Friday 5th
Protein purification of Cas13a Lbu
Protocol: Äkta protein purification
Participants: Ludwig, Chris
Observations:
One test sample was run on the Äkta before the actual run. Only the second run was analyzed with a SDS PAGE.
Results
Protein purification of Cas13a Lsh
Protocol: Äkta protein purification
Participants: Ludwig, Chris
Results
Monday 8th
Transformation of TEV plasmid (form lab in the chemistry department) in DH5α
Protocol: Electro Transformation
Participants: Sven
Tuesday 9th
Dialysis of pooled peak fractions of Cas13a Lbu and Cas13a Lsh into ion exchange buffer
Protocol: Äkta protein purification
Participants: Ludwig, Chris, Sven
PCR of NT and T crDNA (for Cas13a Lbu and Cas13a Lsh)
Protocol:
Participants: Sven
Observations:
Wednesday 10th
Test of different lysis methods for total RNA extraction with Norgen kit
Protocol: Total RNA Purification with Norgen Kit
Participants: Julian, Milica, Jorge
Observations:
Three lysis methods were used: sonifcation, lysozyme digestion and heat lysis in SDS.
Lysozyme digestion: According to protocol.
Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min.
Results
See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17 for gel picture.
Upconcentration fo Cas13a Lbu and Cas13 Lsh after dialysis
Protocol: Äkta protein purification
Participants: Ludwig, Chris
Results
Thursday 11th
Test of different lysis methods for Phenol/Chloroform total RNA extraction.
Protocol: Phenol/Chloroform purification of RNA
Participants: Milica, Sven
Observations:
Three lysis method were used: sonification, lysozyme digestion and heat lysis in SDS.
Lysozyme digestion: according to the total RNA purification with Norgen Kit protocol.
Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min.
The samples were incubated at -80 °C overnight and the purification finished the next day.
Results
See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17.
Phenol/Chloroform purification of RNA from in-vitro transcription
Protocol: Phenol/Chloroform purification of RNA
Participants: Dawafuti, Sven
Observations
The samples were incubated at -80 °C overnight and the purification finished the next day.
Results
Friday 12th
Urea PAGE of in vitro transcription samples
Protocol: UREA PAGE
Participants: Sven
Results
Monday 15th
Preparation fo buffers for Heparin purification of Cas13a Lbu and Cas13a Lsh
Protocol: Äkta protein purification
Participants: Ludwig, Chris
Tuesday 16th
Heparin purification of Cas13a Lbu and Cas13 Lsh
Protocol: Äkta protein purification
Participants: Ludwig, Dawa
Results
Wednesday 17th
Thursday 18th
Friday 19th
Monday 22nd
Tuesday 23rd
PCR of Cas13a Lwa part1 and part2 and backbone pSB1C3_ccdB
Protocol: PCR
Participants: Ludwig, Chris, Rob
Observations:
Q5 polymerase was used
Primers Lwa part1 and part2: VF and VR
Primers pSB1C3_ccdB: p-pSB-GG-Fwd, p-pSB-GG-Rev
Results
Assembly of Cas13a Lwa part1 and part2 with pSB1C3_ccdB using GoldenGate Cloning
Protocol: GoldenGate Assembly
Participants: Ludwig, Chris, Rob
Observations:
Insert to vector ratio: 2:1 and 3:1
Electro Transformation of assembled Lwa parts and pSB1C3 into DH5α and plating on LBCm agar plates
Protocol: Electro Transformation
Participants: Ludwig, Chris, Rob
Observations:
Next day: Only a few colonies.
Electro Transformation of TEV biobricks (pSB1C3-BBa_K1319008 and pSB1C3-BBa_K1319004) and plating on LBCm agar plates
Protocol: Electro Transformation
Participants: Ludwig, Chris, Rob
Wednesday 24th
Repeat: Assembly of Cas13a Lwa part1 and part2 with pSB1C3_ccdB using GoldenGate Cloning
Protocol: GoldenGate Assembly
Participants: Ludwig, Erika, Dawa, Chris
Observations:
Insert to vector ratio: 2:1 and 3:1
Electro Transformation of assembled Lwa parts and pSB1C3 into DH5α and plating on LBCm agar plates
Protocol: Electro Transformation
Participants: Ludwig, Erika, Dawa, Chris
Repeat: PCR of Cas13a Lwa part1 and part2 and backbone pSB1C3_ccdB
Protocol: PCR
Participants: Ludwig, Erika, Dawa, Chris
Observations:
Q5 polymerase was used
Primers Lwa part1 and part2: VF and VR
Primers pSB1C3_ccdB: p-pSB-GG-Fwd, p-pSB-GG-Rev
Results
Preparation of 5 ml LBCm overnight culture of one picked DH5α pSB1C3-Cas13a-LwaLwa colony (from first GoldenGate assembly)
Protocol: -
Participants: Ludwig, Erika, Dawa, Chris
Thursday 25th
Miniprep of DH5α pSB1C3-Cas13a-Lwa overnight culture
Protocol: Minipreparation
Participants: Dawa, Chris
Observations:
pSB1C3-Cas13a-Lwa was then used for an analytical restriction digest and was sent for sequencing (with primer VR)
Analytical restriction digest with pSB1C3-Cas13a-LwaLwa with XbaI (single digest) and XbaI + SpeI (double digest)
Protocol: Restriction digest
Participants: Dawa, Chris
Results
Repeat: PCR of Lwa part2 and also GoldenGate was redone with new amplified Lwa part2
Protocol: PCR and GoldenGate
Participants:
Observations:
PCR Primers: VF, VR
Insert:Vecor 3:1 (GoldenGate)
Results
Friday 26th
Transformation of 1 µl GoldenGate sample in DH5α and plating on LBCm agar plates
Protocol: Transformation
Participants: Ludwig, Dawa, Chris
Observations:
PCR of BBa_K1319004 and BBa_K1319008 (TEV) to check if biobricks are in plasmid
Protocol: PCR
Participants: Ludwig, Dawa, Chris
Results
Electro transformation of BBa_K1319004 and BBa_K1319008 (TEV) in DH5α and plating on LBCm agar plates
Protocol:
Participants:
Observations:
No colonies for BBa_K1319008
Monday 29th
Chemical transformation of BBa_K1319008 (TEV) in DH5α and plating on LBCm agar plates
Protocol: Chemical Transformation
Participants: Ludwig, Dawa, Erika
Observations:
3 µl (= 750 pg) were used
Chemical transformation of GolenGate sample from 24.05. and also from 26.05. in DH5α and plating on LBCm agar plates
Protocol: Chemical Transformation
Participants: Ludwig, Dawa, Erika
Observations:
5 µl for each transformation were used
Tuesday 30th
Colony PCR of DH5α pSB1C3-Cas13a-LwaLwa (from GoldenGate)
Protocol: cPCR
Participants: Chris, Dawa
Results
Colony PCR of DH5α pSB1C3-BBa_K1319004 and pSB1C3-BBa_K1319008 (TEV)
Protocol: cPCR
Participants: Chris, Dawa
Observations:
Primers: VF, VR
Overnight cultures from cultures that showed right cPCR bands were set up
Results
Wednesday 31st
Preparation of buffers for Cas13a purification (Elution, Wash, Ion exchange and SEC)
Protocol: Äkta purification
Participants: Milica, Ludwig, Flo, Sven, Jorge
Observations:
Minipreparation of DH5α pSB1C3-BBa_K1319004 and pSB1C3-BBa_K1319008 (TEV) overnight cultures
Protocol: Miniprep
Participants: Milica, Ludwig, Flo, Sven, Jorge
Observations:
Each construct was sent for sequencing with primers VR and VF
Thursday 1st
Colony PCR of DH5α pSB1C3-Cas13a-Lwa
Protocol: cPCR
Participants: Ludwig, Erika, Sven
Observations:
Results
Äkta purification of Cas13a Lbu and Cas13a Lwa (Affinity chromatography)
Protocol: Äkta protein purification
Participants: Ludwig, Erika, Sven
Results
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
Monday 5th
Preparation of overnight cultures from Rosetta pSB1C3-His-MBP-Lbu-Cas13a-WT and Rosetta p2CT-His-MBP-Lsh-Cas13a-WT glycerol stocks, DH5α pSB-Cas13a-Lwa cPCR colony 5 and
DH5α pSB1C3-BBa_K1319004 cPCR colony 1 and DH5α pSB1C3-BBa_K1319008 cPCR colony 1
Protocol: -
Participants: Sven
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
Inoculation of Rosetta pSB1C3-His-MBP-Lbu-Cas13a-WT and Rosetta p2CT-His-MBP-Lsh-Cas13a-WT overnight cultures in each 1 l LBCarb + Cm medium
Protocol:
Participants: Sven, Patrick
Preparation of glycerol stocks for DH5α pSB1C3-BBa_K1319004 cPCR and DH5α pSB1C3-BBa_K1319008
Protocol: Glycerol cryo stocks
Participants: Sven, Patrick
Minipreparation of DH5α pSB-Cas13a-Lwa cPCR colony 5, DH5α pSB1C3-BBa_K1319004 and DH5α pSB1C3-BBa_K1319008
Protocol: Miniprep
Participants: Sven, Patrick
Wednesday 7th
Harvesting of bacteria with overexpressed Cas13a Lbu and Cas13a Lsh
Protocol: Äkta purification
Participants: Ludwig
Preparation of 5 ml LBCm overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5
Protocol: -
Participants: Jorge
Thursday 8th
Preparation of wash and elution buffer for Cas13a protein purification
Protocol: Äkta protein purification
Participants: Ludwig, Erika
Observations:
pH was checked and adapted after adding of TCEP
Purification of Cas13a Lbu and Cas13a Lsh (Affinity chromatography)
Protocol: Äkta protein purification
Participants: Ludwig, Erika, Sven
Results
Minipreparation of DH5α pSB-Cas13a-Lwa cPCR colony 5 and preparation of glycerol stock
Protocol: Minipreparation and glycerol stock
Participants: Ludwig, Erika, Sven
Friday 9th
PCR of pSB1C3-BBa_K1319008 (TEV) for adding a His tag
Protocol: PCR
Participants: Chris
Observations:
Primers: p-TEV-His-fwd and p-TEV-His-rev
Procedure was follwed by PCR cleanup and DpnI digestion
Results
Monday 12th
Purification of Cas13a Lbu and Cas13a Lsh (Size exclusion chromatography)
Protocol: Äkta purification
Participants: Sven, Ludwig, Benedikt, Milica
Observations:
Cas13a Lbu and Cas13a were upconcentrated to 1 ml each (centrifugal filters: MWCO: 30 kDa).
2 runs for each protein were necessary
Results
PCR of Lwa part2b (reordered gBlock without internal BsaI restriction site)
Protocol:
Participants: Sven, Ludwig, Benedikt, Milica
Observations:
Primers: VF, VR
Procedure was follwed by PCR cleanup
Results
Assembly of Lwa part1, part2b and pSB1C3-ccdB with GoldenGate and transformation in DH5α
Protocol: GoldenGate Cloning
Participants: Sven, Ludwig, Benedikt, Milica
Ligation of pSB1C3-BBa_K1319008 (TEV) after adding the His tag by overhang PCR
Protocol:
Participants: Sven, Ludwig, Benedikt, Milica
Observations:
Procedure was follwed by chemicial transformation in DH5α and plating on LbCm agar plates
Results
Tuesday 13th
Colony PCR of DH5α pSB-Cas13a-Lwa from GoldenGate assembly (with part2b)
Protocol:
Participants: Ludwig, Chris
Results
cPCR showed positive results. Cloning seemed to work, although there was the internal BsaI restriction site
Redone: Transformation of 15 µl ligation sample (of pSB1C3-BBa_K1319008 (TEV) after adding the His tag by overhang PCR)
Protocol:
Participants: Ludwig, Chris
Observations:
Colonies were visible the next day this time. 2 colonies were picked and resuspended in each 5 ml LBCm for overnight cultures
Preparation of 5 ml LBCm overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5
Protocol:
Participants:
Observations:
Inoculation from cyro stock
Results
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
Up-concentration of Cas13a Lbu and Cas13 Lsh after SEC and concentration measurement
Protocol:
Participants: Chris, Patrick
Observations:
Centrifual filters: MWCO: 30 kDa
Concentrations were measured using a Bradford assay
Results
Cas13 Lbu: 746 mg/ml, Cas13 Lsh: 120 mg/ml
Miniprep of overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5
Protocol: Minipreparation
Participants: Chris, Patrick
Observations:
Plasmid was again sent for sequencing with VR and VF
Results
First sequencing looked good
Thursday 15th
Friday 16th
Sending pSB-Cas13a-Lwa cPCR colony 5 for sequencing again with more primers
Protocol: Sequencing
Participants: Chris, Jorge
Observations:
Preparation of first readout experiment with Cas13 Lbu and Cas13 Lsh
Protocol: Readout
Participants: Chris, Jorge
Observations:
Cleavage activity was tested with Aptamers (Malachite, Spinach) and RNase alert
Results
The Cas13a proteins were activated and cleaved the aptamers. This resulted in a decrease in fluorescence intensity.
Lysis-tests with acidic phenol chlorophorm extraction
Protocol: Lysis test pipetting scheme
Protocol: Phenol-Chlorophorm extraction
Participants: Julian, Patrick
Notes:
Cell suspension had OD600 (extrapolated) 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
! Did not calibrate alkaline lysis solution 3 to given pH -> probably more RNA degradation
! Did store RNA at -20 °C instead of -80 -> probably more RNA degradation
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
see also "Denaturing PAGE for ssRNA" Monday, the 26th
Monday 19th
Preparation of 5 ml LBCm overnight culture of DH5α pSB-Cas13a-Lwa cPCR colony 5
Protocol: Overnight culture
Participants: Ludwig
Observations:
Inoculation from cryo stock
Preparation of 5 ml LBCm overnight culture of DH5α pSB1C3-BBa_K1319008-His (TEV) 1 and 2
Protocol: Overnight culture
Participants: Ludwig
Observations:
Inoculation from cryo stock
Electro transformation of pSB1C3-Cas13a-Lwa (cPCR colony 5) into E. coli BL21star
Protocol: Electro transformation
Participants: Ludwig, Chris
Observations:
pSB1C3-Cas13a-Lwa (cPCR colony 5) was verified by sequencing and is named now pSB1C3-Cas13a-Lwa
Tuesday 20th
Minipreparation of DH5α pSB-Cas13a-Lwa cPCR colony 5
Protocol: Minipreparation
Participants: Ludwig, Chris
Observations:
The plasmid was sent for sequencing with VR, VF, p-seq-lwa 1-4
Minipreparation of DH5α pSB1C3-BBa_K1319008-His (TEV) 1 and 2
Protocol: Minipreparation
Participants: Ludwig, Chris
Observations:
The plasmids were sent for sequencing with VR, VF
Preparation of 5 ml LBCm overnight culture of one picked BL21star pSB1C3-Cas13a-Lwa colony
Protocol: Overnight culture
Participants: Ludwig, Chris
Wednesday 21st
Minipreparation of BL21star pSB1C3-Cas13a-Lwa overnight culture and send for sequencing
Protocol: Minipreparation
Participants: Erika, Igor, Milica
Observations:
Primers: VF, VR, p-seq-lwa 1-4
Preparation of glycerol stock for BL21star pSB1C3-Cas13a-Lwa
Protocol: Glycerol stock
Participants: Erika, Igor, Milica
Thursday 22nd
Friday 23rd
Time-series of alkaline lysis, in triplets
Protocol: Lysis test pipetting scheme
Participants: Julian, Patrick
Notes:
Did alkaline Lysis with inbubation/lysis times of 0 min (~10 sec ), 1 min, 3 min, 10 min, Heat+SDS as reference, no Trizol
Cell suspension with OD600 (extrapolated) of 2.182 -> 1.78 x 109 cells/ml
Phenol-Chlorophorm Precipitation at -80 was done until Wednesday
Results
see "Denaturing PAGE for ssRNA" Thursday (not Monday!), the 29th
Monday 26th
Gel analysis of extracted RNA (July 16th !)
Protocol: Denaturing PAGE for ssRNA
Participants: Julian, Patrick
Notes:
Results
Tuesday 27th
Electro Transformation of pSB1C3-BBa_K1319008-His (TEV) in BL21star and plating on LBCm agar plates
Protocol: Electro transformation
Participants: Ludwig, Chris
Observations:
Preparation of 5 ml LBCm + Carb overnight culture of E. coli Rosetta p2CT-His-MBP-Lbu-Cas13a-WT
Protocol: Overnight culture
Participants: Ludwig, Chris
Preparation of 5 ml LBCm overnight culture of E. coli DH5α pSB1C3-BBa_K1319008-His 2
Protocol: Overnight culture
Participants: Ludwig, Chris
Wednesday 28th
Inoculation of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT in 4x 1 l 2xYTCm + Carb medium for expression of Cas13a Lbu
Protocol: Äkta protein purification
Participants: Chris, Ludwig,Jorge
Minipreparation of DH5α pSB1C3-BBa_K1319008-His 2 and send for sequencing
Protocol: Minipreparation and Sequencing
Participants: Chris, Ludwig, Jorge
Observations:
Preparation of 5 ml LBCm overnight culture of BL21star pSB1C3-BBa_K1319008-His from picked colony
Protocol: Overnight culture
Participants: Chris, Ludwig, Jorge
Plating of DH5α pSB1C3-Cas13a-Lwa glycerol stock on LBCm agar plates
Protocol:
Participants:
Observations:
Colonies should be checked if they grow the same and a cPCR should confirm that there is only one plasmid
Thursday 29th
Preparation of glycerol stock of BL21star pSB1C3-BBa_K1319008-His
Protocol: Glycerol stock
Participants: Ludwig, Chris
Observations:
From this glycerol stock a 5 ml LBCm overnight culture was prepared
A 5 ml LBCm overnight culture was prepared directly from this cryo stock
Harvesting of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT with overexpressed Cas13a Lbu
Protocol: Äkta protein purification
Participants: Ludwig, Chris
Observations:
The pellet was stored at -80 °C
Colony PCR of DH5α pSB1C3-Cas13a-Lwa (plated glycerol stock)
Protocol: cPCR
Participants: Ludwig, Chris
Results
Time-series of alkaline lysis, in triplets (continued)
Protocol: Phenol-Chlorophorm extraction
Participants: Patrick
Notes:
Continuing after -80 °C precipitation
Extremely big pellets after 99 % EtOH precipitation for all samples with SDS: Possibly contaminated with DNA
Results
SDS 10⁹ seems to have lower yield than NaOH -> maybe more SDS? (10⁸ has no problems...)
NaOH is done after "0" minutes (about 10 sec for adding base to all eppis and turning 3 times)
Sample
Number of sample
[] [µg/ml]
A260/A280
A260/A230
A230 [A]
A260 [A]
A280 [A]
A320 [A]
1
SDS, 10^8 cells 1 62.4 2.080 0.118 1.343 0.174 0.093 0.018 2
2 56.8 2.029 0.128 1.123 0.153 0.081 0.011 3
3 47.6 1.983 0.102 1.178 0.127 0.068 0.008 4
SDS, 10^9 cells 1 354 1.782 >0.4 >2.5 0.913 0.525 0.029 5
2 260 1.857 0.295 2.222 0.665 0.365 0.015 6
3 376 1.801 0.392 2.422 0.962 0.544 0.022 7
NaOH, 0 min, 10^8 cells 1 39.2 1.690 2.042 0.056 0.106 0.066 0.008 8
2 53.6 1.614 1.887 0.086 0.149 0.098 0.015 9
3 68.8 1.720 2.457 0.070 0.172 0.100 0.000 10
NaOH, 0 min, 10^9 cells 1 629 1.911 2.466 0.643 1.578 0.828 0.005 11
2 17.6 1.692 2.200 0.019 0.043 0.025 -0.001 12
3 619 1.897 2.453 0.636 1.553 0.821 0.005 13
NaOH, 1 min, 10^8 cells 1 52 1.806 2.203 0.059 0.130 0.072 0.000 14
2 33.2 1.729 2.371 0.035 0.083 0.048 0.000 15
3 58.4 1.738 2.393 0.061 0.146 0.084 0.000 16
NaOH, 1 min, 10^9 cells 1 606 1.893 2.469 0.620 1.522 0.807 0.006 17
2 627 1.898 2.431 0.651 1.574 0.832 0.006 18
3 22.4 1.806 2.435 0.023 0.056 0.031 0.000 19
NaOH, 3 min, 10^8 cells 1 44 1.864 2.444 0.044 0.109 0.058 -0.001 20
2 61.2 1.779 2.593 0.058 0.152 0.085 -0.001 21
3 5.6 1.400 2.800 0.004 0.013 0.009 -0.001 22
NaOH, 3 min, 10^9 cells 1 619 1.908 2.432 0.643 1.554 0.818 0.007 23
2 570 1.927 2.468 0.583 1.430 0.745 0.006 24
3 20.4 1.759 2.429 0.020 0.050 0.028 -0.001 25
NaOH, 10 min, 10^8 cells 1 10.8 1.588 2.250 0.012 0.027 0.017 0.000 26
2 8.8 1.571 3.143 0.006 0.021 0.013 -0.001 27
3 4.4 1.375 3.667 0.002 0.010 0.007 -0.001 28
NaOH, 10 min, 10^9 cells 1 503 1.916 2.499 0.508 1.262 0.661 0.005 29
2 564 1.911 2.487 0.572 1.415 0.743 0.005 30
3 611 1.902 2.467 0.625 1.533 0.809 0.006
Friday 30th
Minipreparation of BL21star pSB1C3-BBa_K1319008-His
Protocol: Minipreparation
Participants: Max
Observations:
The plasmid was sent for sequencing with primers VR and VF
Gel analysis of alkaline-extracted RNA time-series (July 23th !)
Protocol: Denaturing PAGE for ssRNA
Participants: Julian, Patrick
Notes:
Results
Monday 3rd
Preparation of 5 ml LBCm overnight culture of BL21star pSB-Cas13a-Lwa
Protocol: Overnight culture
Participants: Max
Tuesday 4th
Inoculation of BL21star pSB-Cas13a-Lwa in 4x 1 l 2xYTCm medium for expression of Cas13a Lwa
Protocol: Ni NTA agarose protein purification
Participants: Max, Dawa
Purification of Cas13a Lbu
Protocol: Ni NTA agarose purification
Participants: Max, Dawa
Observations:
Fresh DTT was added to the lysis buffer
1 ml Ni-NTA agarose was used per 4 ml lysate
Sample was incubated 1 h at 4 °C while shaking
The procedure was followed by TEV cleavage overnigth while dialyzing
Results
Wednesday 5th
Harvesting of BL21star pSB-Cas13a-Lwa with overexpressed Cas13a Lwa
Protocol: Ni NTA protein purification
Participants: Max, Dawa, Erika
Observations:
Pellet was incubated for 1 h at -80 °C before it was used for purification
Results
Purification of Cas13a Lwa
Protocol: Ni NTA agarose purification
Participants: Max, Dawa, Erika
Observations:
Fresh DTT was added to the lysis buffer
1 ml Ni-NTA agarose was used per 4 ml lysate
Sample was incubated 1 h at 4 °C while shaking
Results
Precipitation of TEV plasmid for European meetup in Delft
Protocol: -
Participants: Max, Dawa, Erika
Observations:
pSB1C3-BBa_K1319008-His was lyophylized by Ethanol precipitation and evaporation
Thursday 6th
Friday 7th
Reverse Ni-NTA purification of cut Cas13a Lbu
Protocol: Ni NTA agarose protein purification
Participants: Rob
Observations:
Ni NTA agarose binds the cleaved His-MBP tag, Cas13a Lbu is in the flow-through
Results
Monday 10th
Preparation of 50 ml LBCm overnight culture of BL21star pSB1C3-BBa_K1319008-His (TEV)
Protocol: Overnight culture
Participants: Rob, Ludwig, Rob
Tuesday 11th
Inoculation of BL21star pSB1C3-BBa_K1319008-His (TEV) in 3x 1 l 2xYTCm medium for expression of the TEV protease
Protocol: Äkta protein purification
Participants: Ludwig, Max
Observations:
Induction with 1 mM IPTG at OD600 of 0.6
Expression at 16 °C overnight
RNase alert assay with Cas13a Lbu and Cas13a Lsh
Protocol: Cas13a activity assay
Participants: Igor, Rob
Results
Wednesday 12th
Harvesting of BL21star pSB1C3-BBa_K1319008-His with overexpressed TEV protease
Protocol: Äkta protein purification
Participants: Ludwig, Milica
Thursday 13th
Friday 14th
Protein purification of TEV protease
Protocol: Äkta protein purification
Participants: Ludwig, Max, Sven
Results
Cas13a activity assay with RNase alert
Protocol: Cas13 activity assay
Participants: Dawa, Rob
Results
Monday 17th
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
Observations:
Results
Tuesday 18th
Preparation of 40 ml LBCm + Carb overnight culture of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT
Protocol: Äkta protein purification
Participants: Chris, Max, Dawa
Wednesday 19th
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
Observations:
Results
Inoculation of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT in 3x 1 l 2xYTCm + Carb medium for expression of Cas13a Lbu
Protocol: Äkta protein purification
Participants: Max, Dawa
Thursday 20th
Size exclusion chromatography of affinity purified TEV protease
Protocol: Äkta protein purification
Participants: Max, Sven, Ludwig
Results
Harvesting of Rosetta p2CT-His-MBP-Lbu-Cas13a-WT with overexpressed Cas13 Lbu
Protocol: Äkta protein purification
Participants: Max, Sven, Ludwig
In vitro Transcription of target RNA, Lbu crRNA and Lsh crRNA
Protocol: In vitro Transcription
Participants: Igor
Observations:
Procedure was followed by DNAase treatment and phenol chloroform extraction
Results
Friday 21st
PCR of gBlock of aeBlue
Protocol: PCR
Participants: Chris
Monday 24th
Digestion of insert and pSB1C3 with Pst1-HF and EcoRI-HF
Protocol: Digestion and Ligation
Participants: Chris
Observations:
Plasmid was dephosporylated with arctic phosphatase and insert was ligated with plasmid overnight at 16 °C
Tuesday 25th
Electro transformation of aeBlue ligation sample in E. coli Turbo cells
Protocol: Electro transformation
Participants: Max
Wednesday 26th
Colony PCR of plated pSB1C3-aeBlue
Protocol: cPCR
Participants: Max
Results
Preparation of 5 ml LBCm overnight cultures (picked colonies of aeBlue ligation)
Protocol: Overnight culture
Participants: Max
Thursday 27th
Minipreparation of Turbo pSB1C3-aeBlue overnight cultures and send for sequencing
Protocol: Minipreparation and sequencing
Participants: Max
Friday 28th
Upconcentration of TEV protease to 2 mg/ml and stored in TEV storage buffer
Protocol: Äkta protein purification
Participants: Max
Monday 31th
Tuesday 1st
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.
Thursday 3rd
Friday 4th
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.
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.
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.
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.
Annealing of Invitro Transcription Targe Inhibitor Circuit 3a (IC3a)
Protocol:
Participants: Florian
Observations:
Invitro Transcription of IC3a
Protocol: In vitro transcription
Participants: Florian
Observations:
Incubated over night at 37 ºC
Results
See results of PEC from 11.10.2017
Friday 11th
DNAse I digest and PCE of the RNA IC3a
Protocol: Restriction digest, Phenol-chlorophorm purification of RNA
Participants: Florian
Observations:
Phenol-Chloroform with the wrong pH was used.
Results
Sample was lost due to the use of the wrong Phenol-Chloroform solution.
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
Monday 14th
Invitro Transcription of IC3a
Protocol: In vitro transcription
Participants: Florian
Observations:
Results
See results of Restriction digestion and PCE from 15.08.17
Tuesday 15th
DNAse I digest and PCE of the RNA
Protocol: Restriction digest, Phenol-chlorophorm purification of RNA
Participants: Florian
Observations:
Phenol-Chloroform with the wrong pH was used.
Results
IC3A has a length of 33 bp = band on top.
Wednesday 16th
Thursday 17th
Friday 18th
Sunday 20th
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
Observations:
Results
Monday 21st
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
Observations:
Results
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
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
Observations:
Results
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
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
Observations:
Results
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:
Monday 28th
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
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)
Thursday 31st
RNA extraction from induced Lbu-strain
Protocol: Lysis test pipetting scheme
Protocol: Phenol-Chlorophorm extraction
Protocol: Denaturing PAGE for ssRNA
Participants: Dawa
Notes:
Induced pSB1C3_lbu with IPTG before lysis
Two cultures lysed with SDS + Heat only
No OD600 measurement
Results
See Friday, 1st Sept.
Friday 1st
Intein-Extein-Readout
Protocol: -
Participants: Max
Observations:
Plates showed no colonies, as there was no SOC step for outgrowth
Repeated digestion and Ligation
Ligated 1 C-Terminal and 3 N-Terminal fragments into psb1C3 and psb4A5
RNA extraction from induced Lbu-strain (continued)
Protocol: Denaturing PAGE for ssRNA
Participants: Dawa, Julian
Notes:
Induced pSB1C3_lbu with IPTG before lysis
Two cultures lysed with SDS + Heat only
No OD600 measurement
Results
A
B
C
1
sample conc ~Nanodrop 260/280 2
1 (non-induced) 2192 1.8 3
2 (non-induced) 2320 1.8 4
3 (induced) 508 1.69 5
4 (induced) 442 1.66 6
1,2 showed precipitation of RNA ->measurement/gel wrong if not vortexed completely
A260/280 ration hints to impurities or DNA ->maybe due to too many cells
Saturday 2nd
o
Monday 4th
Total RNA extraction of E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with Norgen kit
Protocol: Total RNA Purification with Norgen Kit
Participants: Jorge, Dawafuti
Observations:
Stored at -80 ºC as TRNA Kit #1-4
Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
Protocol: Protocol
Participants: Jorge, Dawafuti
Observations
Stored at -80 ºC as TRNA PC #1-4.
Results
See results Urea-SDS-PAGE from 05.09.17.
Tuesday 5th
Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT (continued)
Protocol: Denaturing PAGE for ssRNA
Participants: Dawa, Jorge
Notes:
Results
DNA Amplification Test using Recombinase Polymerase Amplification
Protocol: Recombinase Polymerase Amplification
Participants: Sven
Observations:
DNA Amplification with RPA was tested using the provided Control Reaction by TwistDx and our BioBrick His6-TEV plasmid
incubation times of 40 minutes for Control and Sample 1 and 60 minutes for sample 2. Differences due to incubation time
was tested.
RPA according to protocol
PCR cleanup according to protocol.
Results
Positive Control worked. RPA of TEV could not produce the whole (~1300 bp) construct but would produce amplicons of shorter size (100-500 bp)
which was anticipated since TwistDx protocol says optimal amplicon length is 500 bp. Full length double-stranded DNA could consequently not be formed.
Wednesday 6th
Thursday 7th
Friday 8th
Test heat-only lysis (1,3 and 7 minutes))
Protocol: Phenol-Chlorophorm extraction
Participants: Julian
Notes:
Diluted E. coli culture to 10^8 cells/ml, used 300 uL as sample
Heat-only at 93 °C for 15 min, diluted wiht tap water to simulate actual usage scenario (saliva etc..)
Results
Hardly any RNA detectable (concentration < 10 ug/ml). Abysmal 260/280-ratio
-> Heat-only lysis is too inefficient for that low cell counts, use 10^9 next time.
Monday 11th
Tuesday 12th
Wednesday 13th
Heat-lysis with E. coli
Protocol: Phenol-Chlorophorm extraction
Participants: Julian
Notes:
Lysis at 90°C, used 300 uL of E. coli suspension diluted to 2*10^9 cells/ml
SDS-Lysis as reference was impossible with this cell count, interphase during extraction too broad
Results
TODO table
Extraction-yield is down 5- to 10- fold compared to SDS (calculated from SDS-lysis results with 10^9 suspension above)
RPA stability test and Reaction Temperature Screening on paper
Protocol: Recombinase Polymerase Amplification on Paper
Participants: Sven
Observations:
Preparation of stability assay of RPA on paper. Lyophilisation and storage.
RPA on paper according to protocol
Friday 15th
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:
Two samples stored at -80 ºC labeled TRNA Lsh #1 15.09.17 and TRNA Lsh #2 15.09.17
Results
RPA stability test and Reaction Temperature Screening on paper
Protocol: Recombinase Polymerase Amplification on Paper
Participants: Sven
Observations:
Stability of RPA on paper was tested using His6-TEV plasmid after 0, 2 and 24 hours post-lyophilisation. Reaction temperature was tested at 37 °C
as suggested by TwistDx and room temperature. Reaction was positively controlled using non-lyophilised reaction mixture.
RPA according to protocol
PCR cleanup according to protocol.
Results
Lyophilisation of RPA on paper worked. Reaction seems to not be active at room temperature. Directly after lyophilisation, the reaction
efficiency seems to be similar to fresh RPA reaction mixture. However, activity loss is observed already after 2 hours and after 24 hours,
almost no activity is observable anymore.
Monday 18th
Tuesday 19th
Heat-lysis with B. subtilis
Protocol: Phenol-Chlorophorm extraction
Participants: Julian
Notes:
2x SDS-Heat lysis, 2x 90 °C (20 min)
Results
SDS-lysis works a lot worse for gram+ bacteria, Heat-only now better/same (more incubation time...)
Ration relatively bad, probably a lot of cell-wall impurities
TODO table
Wednesday 20th
Thurdsay 21st
Friday 22nd
Monday 25th
Tuesday 26th
Wednesday 27th
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:
Four samples stored at -80 ºC labeled TRNA Lsh #1 27.09.17 and TRNA Lsh #2 27.09.17, TRNA Lsh #3 27.09.17, TRNA Lsh #4 27.09.17.
Results
Thursday 28th
Friday 29th
FINA extraction for RNA with purified total RNA (TRNA Lsh #2-4 from 27.09.17)
Protocol: FINA Extraction for RNA
Participants: Jorge
Observations:
FINA was performed with 25 ul sample instead of 50 ul.
The extraction for TRNA #2 was performed as in protocol.
Before the FINA extraction, TRNA #3 was digested with DNase I according to DNase I Reaction Protocol
After the FINA extraction, the two eluate from TRNA #4 were digested with DNase I according to DNase I Reaction Protocol
First strand cDNA synthesis from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT FINA-RNA eluate
Protocol: First strand cDNA synthesis
Participants: Jorge
Observations
pseq-Lsh-06rev was used as primer.
The eluates from the FINA extractions for RNA from 29.09.17 were used as samples.
From each pair of samples, one was used as a negative control (nuclease free water instead of MuLV-RT).
For all reactions, 6 ul sample were pipetted.
Q5-PCR for Lsh-fragment in E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with cDNA samples from 29.09.17
Protocol: PCR amplification with Q5 Master Mix
Participants: Jorge
Observations:
pseq-Lsh-05fwd was used as forward primer.
pseq-Lsh-06rev was used as reverse primer.
Annealing was performed at 58 ºC.
The amplification step was 110 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
N: FINA extraction performed as in protocol.
NK: FINA extraction as in protocol. No RT in cDNA synthesis (negative control).
P: Digestion of DNA before FINA extraction.
PK: Digestion of DNA before FINA extraction. No RT in cDNA synthesis (negative control).
A: Digestion of DNA after FINA extraction.
AK: Digestion of DNA after FINA extraction. No RT in cDNA synthesis (negative control).
Monday 2nd
Tuesday 3rd
Wednesday 4th
Thursday 5th
In-vitro transcription of AuNP linkers U0, U5, U10, U15
Protocol: in vitro transcription
Participants: Rob
Results
Friday 6th
PCE and gel analysis of of AuNP linkers U0, U5, U10, U15 and new in vitro transcription
Protocol: phenol-chlorophorm purification of RNA
Protocol: Urea gel for RNA detection
Protocol: in vitro transcription
Participants: Rob
Observations:
There was no RNA visible.
The in-vitro transcription was repeated.
Results
Saturday 7th
PCE and gel analysis of of AuNP linkers U0, U5, U10, U15
Protocol: phenol-chlorophorm purification of RNA
Protocol: Urea gel for RNA detection
Protocol: in vitro transcription
Participants: Rob
Observations:
There was no RNA visible.
The in-vitro transcription was repeated with a new batch of T7 RNA polymerase.
Results
See results:
Sunday 8th
PCE and gel analysis of of AuNP linkers U0, U5, U10, U15
Protocol: phenol-chlorophorm purification of RNA
Protocol: Urea gel for RNA detection
Protocol: AuNP preparation
Participants: Rob
Observations:
The IVTX was successful.
3-day incubation of AuNP from stock was started.
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Results
See results from "17-10-8_IVTX_AuNP_linker_U0-U5-U10" and "17-10-8_IVTX_AuNP_linker_U15"
Monday 9th
Wednesday 11th
Cas13a activity assay
Protocol: Cas13a activity assay
Participants: Igor
Observations:
Results
Preparation and DNA-conjugation of of new AuNP
Protocol: AuNP preparation
Protocol: AuNP-DNA conjugation
Participants: Rob
Observations:
Preparation of AuNP was successfully completed.
DNA-conjugation with "AuNP 5' primer" and "AuNP 3' primer"
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Results
See results from "conjugation test"
Thursday 12th
AuNP linkage asssay with DNA-linker
Protocol: AuNP linkage
Participants: Rob
Observations:
1x Buffer was used first, which resulted in no aggregation. Increasing the concentration by adding buffer to a final concentration of 2x afterwards resulted in aggregation and thus was set as the standard.
Results
See results from ""
Friday 13th
AuNP linkage asssay with DNA-linker and varying buffer concentrations
Protocol: AuNP linkage
Participants: Rob
Observations:
2x, 4x, and 6x buffer was used, which resulted in unspecific aggregation from 4x to 6x buffer, thus 2x was kept as standard.
Results
See results from ""
Monday 16th
New DNA-conjugation and AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP linkage
Protocol: AuNP-DNA conjugation
Participants: Rob
Observations:
New AuNP were DNA-conjugated
Water was used instead of linker-RNA.
Results
See results from ""
Tuesday 17th
AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP linkage
Participants: Rob
Observations:
The experiment from the day before resulted in unspecific aggregation in the negative control. To have comparable samples and negative control for the cleavage assay, the experiment was repeated with a mock-DNA in the negative control.
repeated with mock-RNA as negative control. A possible explanation for this is that negatively charched, non-bound RNA in the solution increases repulsion of AuNP.
Results
See results from ""
Wednesday 18th
observation of AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP linkage
Participants: Rob
Observations:
After cooling over night and spinning down for 1 min, just minor aggregation was achieved and samples were kept at 4°C for 2 days to allow for potential further aggregation.
Results
See results from ""
Thursday 19th
Friday 20th
observation of AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP linkage
Participants: Rob
Observations:
The 2-day incubation did not result in higher aggregation, so a new over-night linkage was started.
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After cooling over night and spinning down for 1 min, just minor aggregation was achieved and samples were kept at 4°C for 3 days over the weekend to allow for aggregat
Results
See results from ""
Sunday 22nd
observation of AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP linkage
Participants: Rob
Observations:
After cooling over night and spinning down for 10 min, aggregates could be observed more clearly.
Results
See results from ""
Monday 23rd
Tuesday 24th
observation of AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP-DNA conjugation
Protocol: AuNP linkage
Participants: Rob
Observations:
To have optimal conditions and high DNA density of DNA on the AuNP for linkage and cleavage, a new AuNP-DNA conjugation was performed.
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Over-night linkage was started accoriding to the final protocol.
Results
See results from ""
Wednesday 25th
observation of AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP cleavage
Participants: Rob
Observations:
After spinning down the linked AuNP, all supernatant except for 5 μl was removed, pellet resuspended and spotted on paper.
<\li>
The spotted AuNP were visible as blue aggregates and red, unlinked AuNP.
Results
See results from ""
Thursday 26th
observation of AuNP linkage asssay with RNA-linker and DNA-linker
Protocol: AuNP cleavage
Participants: Rob
Observations:
To assure removal of unbound AuNP from , this time, after spinning down the linked AuNP, all supernatant except for was removed, pellet resuspended in 2 μl 1x Cas13a reaction buffer and spotted on paper.
<\li>
The spotted AuNP were visible as blue aggregates and red, unlinked AuNP.
Results
See results from ""
Friday 27th
Monday 30th
Tuesday 31st
