Difference between revisions of "Team:ETH Zurich/Notebook/Text"

m
Line 7: Line 7:
 
</ul>
 
</ul>
 
</p>
 
</p>
−
07/24/2015&Tab;test
+
 
−
07/25/2015&Tab;test2
+
06/23/2015
 +
<p>
 +
<ul>
 +
<li>Send trafos (bricks and Interlab parts) for sequencing</li>
 +
<li>FACS training</li>
 +
<li>Colony PRC for trafos</li>
 +
<li>Make cryostocks of interlab positive and negative control</li>
 +
<li>Design and order lldRO versions (incl lacO)
 +
</li>
 +
<li>transform again InterLab ligated constructs (twice each)
 +
</li>
 +
</ul>
 +
</p>
 +
 
 +
06/24/2015
 +
<p>
 +
<ul>
 +
<li>Colony PCR for InterLab constructs</li>
 +
<li>Send InterLab constructs for sequencing</li>
 +
<li>Annexin V part : put parts together with backbone
 +
</li>
 +
<li>Amplify lldR from the genome
 +
</li>
 +
<li>Make overnight culture for GFP cells
 +
</li>
 +
<li>Make cryostocks of the 3 constructs (3 replicates)
 +
</li>
 +
<li>Retransform InterLab control I20270?</li>
 +
<li>Design and order all remaining constructs/primers</li>
 +
</ul>
 +
</p>
 +
 
 +
06/25/2015
 +
<p>
 +
<ul>
 +
<li>Got labelled annexin</li>
 +
<li>ON culture for I35104 and the three InterLab promoters (15mL in total) from picked colony
 +
</li>
 +
<li>Run lldR PCR on gel and purify the construct!
 +
</li>
 +
<li> Look into sTRAIL handling (prepare for use)
 +
</li>
 +
<li>GFP competent cells</li>
 +
</ul>
 +
</p>
 +
 
 +
06/26/2015
 +
<p>
 +
<ul>
 +
<li>Miniprep Of InterLab study</li>
 +
<li>Digestion of InterLab-purification</li>
 +
<li>lldP amplification from the genome--> didn't work!</li>
 +
<li>Amplify INP from the biobrick and purify from gel</li>
 +
<li>Alliquoting of TRAIL</li>
 +
</ul>
 +
</p>
 +
 
 +
06/29/2015
 +
<p>
 +
<ul>
 +
<li>Throw away the incorrect parts</li>
 +
<li>Transform TOP10 and gfp strains for efficiency study</li>
 +
<li>Measure concentration of fG0008-fG0012</li>
 +
<li>Ligate InterLab fragments</li>
 +
<li>InterLab transformation</li>
 +
<li>Get Omp-8 cells</li>
 +
<li>TOP10 ON culture</li>
 +
</ul>
 +
</p>
 +
 
 +
06/30/2015
 +
<p>
 +
<ul>
 +
<li> Make TOP10 cryostock</li>
 +
<li>Transformation efficiency study</li>
 +
<li>Colony PCR Interlab</li>
 +
<li>Interlab sent to sequencing</li>
 +
<li>Measure conc of lldP</li>
 +
<li>Transform pSEVA</li>
 +
<li>Make new LB+CM plates</li>
 +
<li>Make an ON culture from c13</li>
 +
</ul>
 +
</p>
 +
 
 +
 
 +
07/01/2015
 +
<p>
 +
<ul>
 +
<li>Passage cells </li>
 +
<li>Pick up Omp-8</li>
 +
<li>Use B0032 (sequenced) for negative control of InterLab</li>
 +
<li>Make new LB</li>
 +
<li>Colony PCR</li>
 +
<li>Send device to sequencing</li>
 +
<li>Omp-8 ON culture at 25ºC</li>
 +
<li>pSEVA ON culture</li>
 +
<li>Competent cells</li>
 +
</ul>
 +
</p>
 +
 
 +
07/02/2015
 +
<p>
 +
<ul>
 +
<li>Miniprep pSEVA</li>
 +
<li>Send one sample of pSEVA for sequencing</li>
 +
<li>Repeat interlab 2 colony PCR (all negative..)</li>
 +
<li>Depending on sequencing results: throw out or correctly label the cryo tube which is now called p32-1</li>
 +
<li>Sample resubmitted for sequencing with reverse primer oiG002</li>
 +
<li>Remake Omp-8 preculture</li>
 +
<li>Make TBF buffer</li>
 +
</ul>
 +
</p>
 +
 
 +
07/03/2015
 +
<p>
 +
<ul>
 +
<li>Mammalian cell passaging</li>
 +
<li>Order oligos</li>
 +
<li>Amplification of annexinV</li>
 +
<li>Digestion of pSEVA low copy</li>
 +
<li>TPR digestion: failed partly</li>
 +
<li>Digest of InterLab 2</li>
 +
</ul>
 +
</p>
 +
 
 +
07/06/2015
 +
<p>
 +
<ul>
 +
<li>Digest pSEVA371</li>
 +
<li>SOC medium</li>
 +
<li>Gibson assembly annexin</li>
 +
<li>Plan mammalian experiments with TRAIL</li>
 +
<li>Transform interlab, annexinV-pSEVA, pSEVA371, pSEVA271</li>
 +
<li>Overnight culture of Omp-8 at 16°, 25°, 37°</li>
 +
<li>Digest pSEVA371</li>
 +
</ul>
 +
</p>
 +
 
 +
07/07/2015
 +
<p>
 +
<ul>
 +
<li>Passage cells</li>
 +
<li>Asked Tania</li>
 +
<li>Comment with Margaux the mammalian experiments</li>
 +
<li>Cryostocks</li>
 +
<li>Primer design</li>
 +
<li>Colombia -> protocol sent</li>
 +
<li>PCR for lldP-lldR</li>
 +
</ul>
 +
</p>
 +
 
 +
07/08/2015
 +
<p>
 +
<ul>
 +
<li>Make LB-agar and LB-medium</li>
 +
<li>pSEVA miniprep</li>
 +
<li>pSEVA digest and purification with miniprep</li>
 +
<li>Purify lldR-lldP fragments (fridge) (digest the template first)</li>
 +
<li>PCR for remaining INP-Annexin fragments</li>
 +
<li>2% agarose gel for remaining INP-Annexin fragments</li>
 +
<li>Make ON cultures of all useful BioBrick transformants for cryo stocks</li>
 +
</ul>
 +
</p>
 +
 
 +
07/09/2015
 +
<p>
 +
<ul>
 +
<li>Start mammalian experiments+ TRAIL
 +
</li>
 +
<li>Shopping list </li>
 +
<li>Colony PCR annexinV colony
 +
</li>
 +
<li>Competent Omp-8
 +
</li>
 +
<li>Mammalian cell FACS</li>
 +
<li>Gibson assembly</li>
 +
<li>Transformation INP-An, lldP-lldR, AnV, p27 and p30</li>
 +
<li>Make LB</li>
 +
<li>Preparation of interlab study</li>
 +
</ul>
 +
</p>
 +
 
 +
07/10/2015
 +
<p>
 +
<ul>
 +
<li>Colony PCR</li>
 +
<li>Cryostocks (still missing 27 and 30)</li>
 +
<li>InterLab FACS done!</li>
 +
<li>Sequencing</li>
 +
<li>Make ITA buffer</li>
 +
</ul>
 +
</p>
 +
 
 +
07/13/2015
 +
<p>
 +
<ul>
 +
<li>Compare results: no results</li>
 +
<li>Colony PCR of lldR-lldP</li>
 +
<li>GA for lldR-lldP diluted</li>
 +
<li>pSEVA271 digestion and miniprep</li>
 +
<li>Preparation of lldR-lldP plates</li>
 +
<li>After meeting planning of experiments</li>
 +
<li>ON culture of AnV and INP-AnV</li>
 +
</ul>
 +
</p>
 +
 
 +
07/14/2015
 +
<p>
 +
<ul>
 +
<li>Cryostock of interlab device 2</li>
 +
<li> lldR-lldP colony PCR</li>
 +
<li>Miniprep of ON of AnV and INP-AnV, send to sequence</li>
 +
<li>TPR re-do</li>
 +
<li>BioBrick assembly of luxI-term</li>
 +
<li>Gel-purify all PCR products from monday</li>
 +
<li>Evening: set up mammalian cells with christian</li>
 +
<li>pSEVA ON for cryostock</li>
 +
<li>BB assembly ligations overnight</li>
 +
</ul>
 +
</p>
 +
 
 +
07/15/2015
 +
<p>
 +
<ul>
 +
<li>Start TRAIL and E Coli mammalian experiments, monitor all day</li>
 +
<li>Colony PCR for new colonies of AnV</li>
 +
<li>ON cultures of AnV</li>
 +
<li>lldR-lldP miniprep</li>
 +
<li>Redo digest for luxI-term assembly (C0161)</li>
 +
<li>Transformation of bb-assembled stuff</li>
 +
<li>Send for sequencing: the "lldR-lldP" plasmid (once with oiG0003 and once with oiG0004)</li>
 +
<li>Make overnight culture from cryostock of INP-Annexin_2 </li>
 +
<li>ON of GFP cells for co-culture</li>
 +
<li>Gibson Assemble Plasmids p62-81</li>
 +
<li>Transformation of gibson assembled plasmids</li>
 +
</ul>
 +
</p>
 +
 
 +
07/16/2015
 +
<p>
 +
<ul>
 +
<li>Make cultures of BB assembled, colony PCR</li>
 +
<li>Make cultures of p62-81, colony PCR, make overnight cultures</li>
 +
<li>Pick up sequencing labes from shop- afternoon (let grow for a long time): miniprep a lot of INP-Annexin, send for sequencing with oiG0004</li>
 +
<li>Omp-8 transformation efficiency</li>
 +
<li>Set up mammalian cells and bacteria for lactate experiment</li>
 +
<li>Mini-prep C0161 and B0012</li>
 +
<li>Digest C0161 and B0012</li>
 +
<li>Overnight culture for interlab</li>
 +
</ul>
 +
</p>
 +
 
 +
07/17/2015
 +
<p>
 +
<ul>
 +
<li>Set up reader plate</li>
 +
<li>Gibson assembly for lldR w/o lldP</li>
 +
<li>Transform and plate lldR constructs</li>
 +
<li>Lactate experiment: change medium at 12:30</li>
 +
<li>Make cryostocks of all things to sequence (annexin and all assemblies)</li>
 +
<li>Send all things for sequencing </li>
 +
<li>Use TOP10 culture for lactate experiment</li>
 +
<li>Repeat co-culture 14:00-16:00</li>
 +
<li>Redo digest of C0161 and B0012 (includde test of enzymes)</li>
 +
<li></li>
 +
</ul>
 +
</p>
 +
 
 +
07/19/2015
 +
<p>
 +
<ul>
 +
<li>Set up overnight cultures of cryos made on friday plus maybe more of each assembly from 0715 plus from assembly on 0717</li>
 +
</ul>
 +
</p>
 +
-
 +
 
 +
07/20/2015
 +
<p>
 +
<ul>
 +
<li>Check sequencing results</li>
 +
<li>Data process: lactate</li>
 +
<li>Redo digest to test enzymes</li>
 +
<li>Send stuff to sequence</li>
 +
<li>New fragments with new fragments</li>
 +
<li>Make colony PCR</li>
 +
<li>Throw away stuff from cryostocks</li>
 +
<li>Make overnight cultures of what is missing: (p72 and p73)</li>
 +
<li>Plan mammalian experiments to know how many cells we need each day
 +
</li>
 +
<li>Process data (FACS, InterLab and lactate)
 +
</li>
 +
<li>Make o/n cultures for interlab in triplicates
 +
</li>
 +
</ul>
 +
</p>
 +
 
 +
07/21/2015
 +
<p>
 +
<ul>
 +
<li>Early do interlab and InterLab sumitted</li>
 +
<li>Keep making new fragments with new primers</li>
 +
<li>If sequencing is correct, make fragments for PL(5)</li>
 +
<li>Send p72 to sequence</li>
 +
<li>New fragments: DPN1 digest, gel, purify from gel</li>
 +
<li>Miniprep stuff</li>
 +
<li>Continue TPR, ligate and transform</li>
 +
</ul>
 +
</p>
 +
 
 +
07/22/2015
 +
<p>
 +
<ul>
 +
<li>TRAIL experiment start at 9:00 fro 3T3</li>
 +
<li>Repeat transformation of lldR and TPR</li>
 +
<li>Miniprep c44 and c45 and INP-Ann and p72 to have more plasmid
 +
</li>
 +
<li>Sample organisation study
 +
</li>
 +
<li>Colony PCR and make ON culture of lldR andTRP(12_114_34)</li>
 +
<li>Send 50 for sequencing with different primer</li>
 +
<li>Miniprep p73 variants for sequencing</li>
 +
<li>Colony PCR</li>
 +
<li>TRAIL experiment 15:30-17:00 FACS machine</li>
 +
<li>Continue TPR assembly
 +
</li>
 +
<li>Digest BioBrick backbone
 +
</li>
 +
<li>Annexin buffer binding made new</li>
 +
<li>TOP10 ON</li>
 +
<li></li>
 +
</ul>
 +
</p>
 +
 
 +
07/23/2015
 +
<p>
 +
<ul>
 +
<li>Colony PCR of GFP-INP-Annexin</li>
 +
<li>Colony PCR of lldR, 12_117, TPR</li>
 +
<li>Make more LB</li>
 +
<li>Digestion Annexin- bb--> no band on annexin</li>
 +
<li>Send TPR for sequencing</li>
 +
<li>ON culture of AnnexinV and B0012</li>
 +
<li>Making Competent Top10 cells</li>
 +
</ul>
 +
</p>

Revision as of 14:46, 24 July 2015

06/22/2015

  • Interlab study:ligation and transformation
  • Redo transformation that failed
  • Order primers, Antibodies and lactate kits

06/23/2015

  • Send trafos (bricks and Interlab parts) for sequencing
  • FACS training
  • Colony PRC for trafos
  • Make cryostocks of interlab positive and negative control
  • Design and order lldRO versions (incl lacO)
  • transform again InterLab ligated constructs (twice each)

06/24/2015

  • Colony PCR for InterLab constructs
  • Send InterLab constructs for sequencing
  • Annexin V part : put parts together with backbone
  • Amplify lldR from the genome
  • Make overnight culture for GFP cells
  • Make cryostocks of the 3 constructs (3 replicates)
  • Retransform InterLab control I20270?
  • Design and order all remaining constructs/primers

06/25/2015

  • Got labelled annexin
  • ON culture for I35104 and the three InterLab promoters (15mL in total) from picked colony
  • Run lldR PCR on gel and purify the construct!
  • Look into sTRAIL handling (prepare for use)
  • GFP competent cells

06/26/2015

  • Miniprep Of InterLab study
  • Digestion of InterLab-purification
  • lldP amplification from the genome--> didn't work!
  • Amplify INP from the biobrick and purify from gel
  • Alliquoting of TRAIL

06/29/2015

  • Throw away the incorrect parts
  • Transform TOP10 and gfp strains for efficiency study
  • Measure concentration of fG0008-fG0012
  • Ligate InterLab fragments
  • InterLab transformation
  • Get Omp-8 cells
  • TOP10 ON culture

06/30/2015

  • Make TOP10 cryostock
  • Transformation efficiency study
  • Colony PCR Interlab
  • Interlab sent to sequencing
  • Measure conc of lldP
  • Transform pSEVA
  • Make new LB+CM plates
  • Make an ON culture from c13


07/01/2015

  • Passage cells
  • Pick up Omp-8
  • Use B0032 (sequenced) for negative control of InterLab
  • Make new LB
  • Colony PCR
  • Send device to sequencing
  • Omp-8 ON culture at 25ºC
  • pSEVA ON culture
  • Competent cells

07/02/2015

  • Miniprep pSEVA
  • Send one sample of pSEVA for sequencing
  • Repeat interlab 2 colony PCR (all negative..)
  • Depending on sequencing results: throw out or correctly label the cryo tube which is now called p32-1
  • Sample resubmitted for sequencing with reverse primer oiG002
  • Remake Omp-8 preculture
  • Make TBF buffer

07/03/2015

  • Mammalian cell passaging
  • Order oligos
  • Amplification of annexinV
  • Digestion of pSEVA low copy
  • TPR digestion: failed partly
  • Digest of InterLab 2

07/06/2015

  • Digest pSEVA371
  • SOC medium
  • Gibson assembly annexin
  • Plan mammalian experiments with TRAIL
  • Transform interlab, annexinV-pSEVA, pSEVA371, pSEVA271
  • Overnight culture of Omp-8 at 16°, 25°, 37°
  • Digest pSEVA371

07/07/2015

  • Passage cells
  • Asked Tania
  • Comment with Margaux the mammalian experiments
  • Cryostocks
  • Primer design
  • Colombia -> protocol sent
  • PCR for lldP-lldR

07/08/2015

  • Make LB-agar and LB-medium
  • pSEVA miniprep
  • pSEVA digest and purification with miniprep
  • Purify lldR-lldP fragments (fridge) (digest the template first)
  • PCR for remaining INP-Annexin fragments
  • 2% agarose gel for remaining INP-Annexin fragments
  • Make ON cultures of all useful BioBrick transformants for cryo stocks

07/09/2015

  • Start mammalian experiments+ TRAIL
  • Shopping list
  • Colony PCR annexinV colony
  • Competent Omp-8
  • Mammalian cell FACS
  • Gibson assembly
  • Transformation INP-An, lldP-lldR, AnV, p27 and p30
  • Make LB
  • Preparation of interlab study

07/10/2015

  • Colony PCR
  • Cryostocks (still missing 27 and 30)
  • InterLab FACS done!
  • Sequencing
  • Make ITA buffer

07/13/2015

  • Compare results: no results
  • Colony PCR of lldR-lldP
  • GA for lldR-lldP diluted
  • pSEVA271 digestion and miniprep
  • Preparation of lldR-lldP plates
  • After meeting planning of experiments
  • ON culture of AnV and INP-AnV

07/14/2015

  • Cryostock of interlab device 2
  • lldR-lldP colony PCR
  • Miniprep of ON of AnV and INP-AnV, send to sequence
  • TPR re-do
  • BioBrick assembly of luxI-term
  • Gel-purify all PCR products from monday
  • Evening: set up mammalian cells with christian
  • pSEVA ON for cryostock
  • BB assembly ligations overnight

07/15/2015

  • Start TRAIL and E Coli mammalian experiments, monitor all day
  • Colony PCR for new colonies of AnV
  • ON cultures of AnV
  • lldR-lldP miniprep
  • Redo digest for luxI-term assembly (C0161)
  • Transformation of bb-assembled stuff
  • Send for sequencing: the "lldR-lldP" plasmid (once with oiG0003 and once with oiG0004)
  • Make overnight culture from cryostock of INP-Annexin_2
  • ON of GFP cells for co-culture
  • Gibson Assemble Plasmids p62-81
  • Transformation of gibson assembled plasmids

07/16/2015

  • Make cultures of BB assembled, colony PCR
  • Make cultures of p62-81, colony PCR, make overnight cultures
  • Pick up sequencing labes from shop- afternoon (let grow for a long time): miniprep a lot of INP-Annexin, send for sequencing with oiG0004
  • Omp-8 transformation efficiency
  • Set up mammalian cells and bacteria for lactate experiment
  • Mini-prep C0161 and B0012
  • Digest C0161 and B0012
  • Overnight culture for interlab

07/17/2015

  • Set up reader plate
  • Gibson assembly for lldR w/o lldP
  • Transform and plate lldR constructs
  • Lactate experiment: change medium at 12:30
  • Make cryostocks of all things to sequence (annexin and all assemblies)
  • Send all things for sequencing
  • Use TOP10 culture for lactate experiment
  • Repeat co-culture 14:00-16:00
  • Redo digest of C0161 and B0012 (includde test of enzymes)

07/19/2015

  • Set up overnight cultures of cryos made on friday plus maybe more of each assembly from 0715 plus from assembly on 0717

-

07/20/2015

  • Check sequencing results
  • Data process: lactate
  • Redo digest to test enzymes
  • Send stuff to sequence
  • New fragments with new fragments
  • Make colony PCR
  • Throw away stuff from cryostocks
  • Make overnight cultures of what is missing: (p72 and p73)
  • Plan mammalian experiments to know how many cells we need each day
  • Process data (FACS, InterLab and lactate)
  • Make o/n cultures for interlab in triplicates

07/21/2015

  • Early do interlab and InterLab sumitted
  • Keep making new fragments with new primers
  • If sequencing is correct, make fragments for PL(5)
  • Send p72 to sequence
  • New fragments: DPN1 digest, gel, purify from gel
  • Miniprep stuff
  • Continue TPR, ligate and transform

07/22/2015

  • TRAIL experiment start at 9:00 fro 3T3
  • Repeat transformation of lldR and TPR
  • Miniprep c44 and c45 and INP-Ann and p72 to have more plasmid
  • Sample organisation study
  • Colony PCR and make ON culture of lldR andTRP(12_114_34)
  • Send 50 for sequencing with different primer
  • Miniprep p73 variants for sequencing
  • Colony PCR
  • TRAIL experiment 15:30-17:00 FACS machine
  • Continue TPR assembly
  • Digest BioBrick backbone
  • Annexin buffer binding made new
  • TOP10 ON

07/23/2015

  • Colony PCR of GFP-INP-Annexin
  • Colony PCR of lldR, 12_117, TPR
  • Make more LB
  • Digestion Annexin- bb--> no band on annexin
  • Send TPR for sequencing
  • ON culture of AnnexinV and B0012
  • Making Competent Top10 cells