Difference between revisions of "Team:Munich/LabJournal"

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<div class="month" id="October" >
 
<div class="month" id="October" >
 
    <div class="date fistOfMonth">Monday 2nd</div>
 
    <div class="date fistOfMonth">Monday 2nd</div>
     
+
    <ul class="myEntry readout">
 +
    <li class="myTitel">
 +
    <b>Live Invitro Transcription test with LBU and Inhibitor-RNA/Activator-DNA dimer with different T7 concentration</b>
 +
    </li>
 +
    <li class="myProtocol">
 +
                                      Protocol: <a href="/Team:Munich/Protocols">In vitro transcription</a>
 +
    </li>
 +
    <li class="myParticipants">
 +
    Participants: Florian
 +
    </li>
 +
    <li class="myObservations">
 +
    Observations:
 +
 
 +
    <img src="https://static.igem.org/mediawiki/2017/9/9a/T--Munich--Results_20171002_live_invitro_trasncription_scheme.png">
 +
                                          <ul>
 +
                                              <li>Forgot to add C3ds with single strand overhang, which is needed for activation of invitro transcription after binding of DNA oligo C3a (activator of C3ds).</li>
 +
                                          </ul>
 +
 
 +
    </li>
 +
    <li class="myResults">
 +
                                          <h4>Results</h4>
 +
                                          <ul>
 +
                                              <li>Plate reader data showed no activity for all samples, as expected without DNA template to transcribe.</li>
 +
                                          </ul>
 +
    </li>
 +
    </ul>    
 
    <div class="date">Tuesday 3rd</div>
 
    <div class="date">Tuesday 3rd</div>
 
     
 
     

Revision as of 09:11, 28 October 2017


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.

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

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:
    • PCR didn't work.
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
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

    xxxChange
  • 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

    xxxChange
  • 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:
    • No colonies visible
  • 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:
    • Primers: VR, VF
  • 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

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

    xxxChange
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

    xxxChange
  • 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
    MethodSDSNaOHTRIzol
    2
    prepare1 mL TES (10mM Tris-HCl, 10mM EDTA, pH 8.0, 2% SDS -> TE-Buffer + SDS...
    3
    ----Lysis1x3x1x
    4
    separate to tubes Spin 300xG, 5 min ->Ice, TROzol to -20! (100 miL at OD 1 -> 10^7 cells, Max. according to TRIzolPaper100 miL bactSusp100 miL bact-susp100 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, vortexone probe after another
    7
    (Addition of inhibitor); Transform total volume to gel tuberesuspend 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:
    • Primer: p-seq-lwa 1-4
  • 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.
  • xxxChange
  • 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 cells36.8551.7042.0440.0470.0940.0560.002
    2
    SDS, 10^9 cells*1193571.6932.0690.1460.3000.1780.002
    3
    NaOH, 0 min, 10^8 cells35.2531.7252.1460.0420.0890.0520.001
    4
    NaOH, 0 min, 10^9 cells5868791.9231.9930.7401.4700.7670.005
    5
    NaOH, 1 min, 10^8 cells73.61101.2350.5860.3170.1870.1520.003
    6
    NaOH, 1 min, 10^9 cells3705551.9451.9580.4750.9270.4780.003
    7
    NaOH, 3 min, 10^8 cells*2593891.8491.4910.4390.6520.3550.005
    8
    NaOH, 3 min, 10^9 cells*2754131.8502.2120.3160.6930.3680.005
    9
    TRIzol, 10^8 cells1942651.3620.2212.2000.4870.3580.002
    10
    TRIzol, 10^9 cells1602181.4290.4880.8220.4030.2830.003
  • Measured [RNA] by Implen Nanophotometer (20170616).
  • **adjusted for different supernatant V aspirated at extraction step
  • Barplots of measure RNA concentrations
  • 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:
    • see Friday, 16th
  • Results

    Gel with degraded RNA
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:
    • 1 µl was used
  • 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:
    • Primers: VF, VR
  • 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 cells162.42.0800.1181.3430.1740.0930.018
      2
      256.82.0290.1281.1230.1530.0810.011
      3
      347.61.9830.1021.1780.1270.0680.008
      4
      SDS, 10^9 cells13541.782>0.4>2.50.9130.5250.029
      5
      22601.8570.2952.2220.6650.3650.015
      6
      33761.8010.3922.4220.9620.5440.022
      7
      NaOH, 0 min, 10^8 cells139.21.6902.0420.0560.1060.0660.008
      8
      253.61.6141.8870.0860.1490.0980.015
      9
      368.81.7202.4570.0700.1720.1000.000
      10
      NaOH, 0 min, 10^9 cells16291.9112.4660.6431.5780.8280.005
      11
      217.61.6922.2000.0190.0430.025-0.001
      12
      36191.8972.4530.6361.5530.8210.005
      13
      NaOH, 1 min, 10^8 cells1521.8062.2030.0590.1300.0720.000
      14
      233.21.7292.3710.0350.0830.0480.000
      15
      358.41.7382.3930.0610.1460.0840.000
      16
      NaOH, 1 min, 10^9 cells16061.8932.4690.6201.5220.8070.006
      17
      26271.8982.4310.6511.5740.8320.006
      18
      322.41.8062.4350.0230.0560.0310.000
      19
      NaOH, 3 min, 10^8 cells1441.8642.4440.0440.1090.058-0.001
      20
      261.21.7792.5930.0580.1520.085-0.001
      21
      35.61.4002.8000.0040.0130.009-0.001
      22
      NaOH, 3 min, 10^9 cells16191.9082.4320.6431.5540.8180.007
      23
      25701.9272.4680.5831.4300.7450.006
      24
      320.41.7592.4290.0200.0500.028-0.001
      25
      NaOH, 10 min, 10^8 cells110.81.5882.2500.0120.0270.0170.000
      26
      28.81.5713.1430.0060.0210.013-0.001
      27
      34.41.3753.6670.0020.0100.007-0.001
      28
      NaOH, 10 min, 10^9 cells15031.9162.4990.5081.2620.6610.005
      29
      25641.9112.4870.5721.4150.7430.005
      30
      36111.9022.4670.6251.5330.8090.006
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:
    • see Friday, 23th
  • Results

    Gel RNA degrading with lysis time
Monday 3rd
  • Preparation of 5 ml LBCm overnight culture of BL21star pSB-Cas13a-Lwa
  • Protocol: Overnight culture
  • Participants: Max
Tuesday 4th
  • 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

    xxxChange
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

    xxxChange
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

    xxxChange
Wednesday 12th
  • Harvesting of BL21star pSB1C3-BBa_K1319008-His with overexpressed TEV protease
  • Protocol: Äkta protein purification
  • Participants: Ludwig, Milica
Thursday 13th
Friday 14th
  • Cas13a activity assay with RNase alert
  • Protocol: Cas13 activity assay
  • Participants: Dawa, Rob
  • Results

    xxxChange
Monday 17th
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
  • 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

    xxxChange
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

    xxxChange
  • Preparation of 5 ml LBCm overnight cultures (picked colonies of aeBlue ligation)
  • Protocol: Overnight culture
  • Participants: Max
Thursday 27th
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
  • 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
Wednesday 16th
Thursday 17th
  • DNA RNA Hybrid Annealing of IC3A with Circuit 3 Activator (C3A) – IC3a concentration screen
  • Protocol:
  • Participants: Florian
  • Observations:
    • Incubation at room temperature for 1 h of DNA and RNA with varied concentrations of IC3a (RNA).
  • Results

    • 4% Agarose does not seem to work for separation. No oligos at all visible after staining with SybrGreen II and SybrGold.
Friday 18th
Sunday 20th
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
  • DNA-RNA-Hybrid-Building
  • Protocol:
  • Participants: Florian
  • Observations:
  • Native PAGE for short DNA oligos
  • Protocol:
  • Participants: Florian
  • Observations:
  • Results

    • Slight band shift of the oligo between 4 and 5 µM.
    • Slight band shift of the oligo between 4 and 5 µM.
    • Looks better than 30% PAGE in its resolution of the DNA oligos.
  • 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:
    • See entry from 24.08.17
Monday 28th
  • DNA RNA Hybrid Annealing of IC3A with Circuit 3 Activator (C3A) – IC3A concentration screen (high concentration)
  • Protocol:
  • Participants: Florian
  • Observations:
  • Results

    • Native PAGE 20 %
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
  • DNA and RNA hybrid of 3C-A and IC3-A
  • Protocol:
  • Participants: Florian
  • Observations:
    • Concentration screen IC3a between 1 and 5 µM. Incubation for dimerization again for 1 hour and at room temperature.
  • Results

    • 20% Native PAGE, 12.5 mM MgCl2
    • Shift of DNA-oligo band with higher concentration of IC3a RNA until 3.5 µM. No band of single DNA oligo C3a at 5 µM visible anymore. Hypothesis of full dimerization, but with a big background of single stranded IC3a RNA.
  • 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
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
      sampleconc ~Nanodrop260/280
      2
      1 (non-induced)21921.8
      3
      2 (non-induced)23201.8
      4
      3 (induced)5081.69
      5
      4 (induced)4421.66
      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
  • Invitro Transcription IC3a RNA
  • Protocol: In vitro transcription
  • Participants: Florian
  • Observations:
  • Results

    See results of DNAseI digestion and PCE from 06.09.17
  • 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:
    • Ran urea gel
  • Results

    20170905_rna_1-8_Dawa_9_12_Jul.Tif
  • 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
  • First try of Cas13a digestion of IC3a/C3a dimer over 1 hour
  • Protocol: RNAse activity protocol
  • Participants: Florian
  • Observations:
    • Incubation for dimerization at room temperature for 1 h. Afterwards 1 hour digestion with Cas13a.
  • Results

    • No real difference visible after digestion for 1 h.
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
  • Second try of Cas13a digestion of IC3a/C3a dimer for several hours and overnight
  • Protocol: RNAse activity protocol
  • Participants: Florian
  • Observations:
  • Results

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.
    Gel Picture 20170915_Sven_RPA_time_stability
Monday 18th
Tuesday 19th
  • IC3a invitro transcription
  • Protocol: In vitro transcription
  • Participants: Florian
  • Observations:
  • Results

    See results of DNaseI digestion and PCE from 20.09.17.
  • 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
  • RNA/DNA hybrid dimerization with Cy5 DNA-oligo C3a
  • Protocol:
  • Participants: Florian
  • Observations:
  • Results

    • Native PAGE 20%, 12.5 mM MgCl2
    • C3a too high concentrated (Red bands). 5 µM instead of 500 nM. Better results with band shift see 29-09-2017.
  • Screen of LBU activity on RNA/DNA dimer
  • Protocol: RNAse activity protocol
  • Participants: Florian
  • Observations:
  • Results

    • Native PAGE 20%, 12.5 mM MgCl2
    • No visible shift of activator DNA oligo with addition of RNA under the use of Cy5 modified DNA activator oligo. Results are bad because of too high C3A DNA oligo concentration. (C3a = red bands).
Friday 29th
  • DNA and RNA hybrid of 3C-A and IC3-A
  • Protocol:
  • Participants: Florian
  • Observations:
  • Results

    • Native PAGE 20%, 12.5 mM MgCl2
    • Visible small up shift of RNA/DNA dimer versus samples, which only contain the DNA oligo by itself. (DNA oligo C3a marked with Cy5 visible as red bands.)
  • 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
  • Live Invitro Transcription test with LBU and Inhibitor-RNA/Activator-DNA dimer with different T7 concentration
  • Protocol: In vitro transcription
  • Participants: Florian
  • Observations:
    • Forgot to add C3ds with single strand overhang, which is needed for activation of invitro transcription after binding of DNA oligo C3a (activator of C3ds).
  • Results

    • Plate reader data showed no activity for all samples, as expected without DNA template to transcribe.
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
Saturday 7th
Sunday 8th
Monday 9th
Wednesday 11th
  • 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" <\li>
  • 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. <\li>
    • 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. <\li>
    • 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