Difference between revisions of "Team:ETH Zurich/Notebook/Text"
Line 534: | Line 534: | ||
<li>Overnight cultures of INP-annexins and c89-c98</li> | <li>Overnight cultures of INP-annexins and c89-c98</li> | ||
<li>Lactate experiment e coli</li> | <li>Lactate experiment e coli</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 11/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Cryostocks PL3, Pl4, lldr, miniprep, sent for sequencing</li> | ||
+ | <li>Colony PCR PL9</li> | ||
+ | <li>Lactate experiment with bacteria twice</li> | ||
+ | <li>ON of pseva271</li> | ||
+ | <li>Colony PCR of PL(9), make ON if it worked (didn't work)</li> | ||
+ | <li>Throw away not confirmed cryos</li> | ||
+ | <li>Redo PL9 assembly and trafo</li> | ||
+ | <li>Do new assemblies with just arrived primers</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 12/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Miniprep and digest bb</li> | ||
+ | <li>PL4 and PL3-3 sent for sequencing</li> | ||
+ | <li>Colony PCR of PL(9), didn't work</li> | ||
+ | <li>Redo assembly for PL(9) with different backbones and transform</li> | ||
+ | <li>Assemble and transform lldR-lldP, annexinseva, annexinbb, inpyfp</li> | ||
+ | <li>TRAIL experiment</li> | ||
+ | <li>Lactate addition experiment with bacteria twice</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | |||
+ | 13/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Colony PCR</li> | ||
+ | <li>Replan mammalian cell experiment: why TRAIL is not working?</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 14/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Cryostocks of new cultures</li> | ||
+ | <li>Colony PCR of PL9</li> | ||
+ | <li>Miniprep of lldrp, pl9, pl3, pl4 and AnnBBBB and sent some for sequencing</li> | ||
+ | <li>Miniprep of lldrp, pl9, pl3, pl4 and AnnBBBB and sent some for sequencing</li> | ||
+ | <li>Preparation of apoptotic cells (TRAIL)</li> | ||
+ | <li>Co-culture HL-60 and bacteria</li> | ||
+ | <li>Split cells</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 15/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Lactate dose-response experiment with bacteria</li> | ||
+ | <li>Darmstadt visit</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 16/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Lactate dose-response experiment with bacteria</li> | ||
+ | <li>Darmstadt visit</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 17/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Send for sequencing lldrp, pl9 etc</li> | ||
+ | <li>Good ones PL3 measured at high conc</li> | ||
+ | <li>Bad ones (65-68) PL(3) measured at same conc</li> | ||
+ | <li>Microscopy TRAIL</li> | ||
+ | <li>Miniprep</li> | ||
+ | <li>Repeat lactate measurement for cells at 6h</li> | ||
+ | <li>Make overnight cultures of PL(3) versions, top10, lldr, lldplldr</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 18/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Bad ones of PL(3) in plate reader at 25mM, 6.3, 3.1, 390uM, 100uM, 10uM</li> | ||
+ | <li>Make fragments from PL(9)</li> | ||
+ | <li>Measure lactate from mammalian cells from yesterday (morning before other exp.)</li> | ||
+ | <li>Make competent cells</li> | ||
+ | <li>Good ones again with high conc at gain 70</li> | ||
+ | <li>More minipreps</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | 19/08/15 | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Remaining bad ones of PL(3) in plate reader all conc | ||
+ | </li> | ||
+ | <li>Send things for sequencing</li> | ||
+ | <li>Remake fragment f48 failed</li> | ||
+ | <li>Pick up trihydrochlorine</li> | ||
</ul> | </ul> | ||
</p> | </p> |
Revision as of 08:40, 20 August 2015
22/06/2015
- Interlab study:ligation and transformation
- Redo transformation that failed
- Order primers, Antibodies and lactate kits
23/06/2015
- 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)
24/06/2015
- 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
25/06/2015
- 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
26/06/2015
- 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
29/06/2015
- 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
30/06/2015
- 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
01/07/2015
- 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
02/07/2015
- 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
03/07/2015
- Mammalian cell passaging
- Order oligos
- Amplification of annexinV
- Digestion of pSEVA low copy
- TPR digestion: failed partly
- Digest of InterLab 2
06/07/2015
- 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
08/07/2015
- 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
09/07/2015
- 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
10/07/2015
- Colony PCR
- Cryostocks (still missing 27 and 30)
- InterLab FACS done!
- Sequencing
- Make ITA buffer
13/07/2015
- 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
14/07/2015
- 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
15/07/2015
- 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
16/07/2015
- 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
17/07/2015
- 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)
19/07/2015
- Set up overnight cultures of cryos made on friday plus maybe more of each assembly from 0715 plus from assembly on 0717
20/07/2015
- 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
21/07/2015
- 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
22/07/2015
- 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
23/07/2015
- 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
24/07/2015
- Miniprep annexin
- Digest INP-An, B0012
- Retransform TPR minipreped yesterday
- Amplify TRP to use in assemblies if sequencing is correct
- Send a lot of things for sequencing (lldR, 12_117)
- Do gibson assembly for pl5, transform
26/07/2015
- Precultures
27/07/2015
- Experiment with Jurkat cells started (lactate)
- Ligation AnV-BB
- Ordered new lldR-lldP plasmid gblocks
- Miniprep ON cultures (TPR and PL(5))
- Send ON cultures (TPR and PL(5))for sequencing
28/07/15
- Make more TAE
- Miniprep lldR ON cultures
- Send lldR for sequencing
- lldR gibson assembly
- lldR transformation
- ON cultures of AnV(c73), INP-AnV(c55), PL(5)(cryos 2b, 1, 2a), PL(7), TOP10 cells with no plasmid (c13)
29/07/15
- Book plate reader
- Think about PS-coated
- ON Cultures annexin in BBBB
- Experiment Annexin-INP in the membrane
- Order one TPR in IDT
30/07/15
- Digest of INP-Annexin (failed)
- Digest of luxI and B0012 (failed)
- ON cultures pSEVA 271 and 371
- ON cultures of B0012 (c27), INP-AnV, luxI
- AnV in BioBrick BackBone sent to sequencing
- Western blot for annexin-inp
- Make ON cultures from p101 (lldR-lldP) plates
- Make new LB and LB-agar
- ON cultures of PL(6)
- Mammalian set up
31/07/15
- Miniprep pSEVA
- Miniprep PL6
- Miniprep and digest of B0012 (c27), INP-AnV and luxI and ligate
- TRAIL jurkat and 3t3
- Lactate all lines + calibration curve
- PCR for fragments 74-80, 97, 98
- Digest test
01/08/15
- Gibson assembly lldP-lldR
- Transformation
02/08/15
- Colony PCR of up to 20 colonies with primers oi3 and oi19
- Make ON cultures, two of every positive colony from the PCR
03/08/15
- Order gblock
- Track selection
- Cryostocks and miniprep lldR-lldP
- Sent stuff for sequencing
- Lactate evaluation (FAIL)
- Try PCR (p24 + o61/o62 = f64) with Q5 2x Master mix (783bp fragment) at 62, 64 and 66°C (FAIL)
- CRYO STOCKS!!!! miniprep and send for sequencing: lldR-lldP
- 4pm meeting doctor
- Order primers
04/08/15
- Start cultures of lldR and lldR-lldP for electrocompetent cells (let grow to OD 0.6)
- Prepare everything for lactate based experiments
- cultures of backbone 371
- order cryotubes
- transform pl3 into intermediate plasmid strain
- make gibson assembly again and transform
05/08/15
- Crystock of pSEVA
- Miniprep pSEVA+ failed digest
- Colony PCR of PL3
- GA for lldP-lldR (old-old one-with low concentration-): one 1h 45ºC, the other 2h 50ºC
- Seeding new mammalian cell line
- ON culture of pSEVA
- Redo Gibson Assembly of p64 and p73, transform and incubate o/n
07/08/15
- Miniprep p50
- Assemble lldPR, PL3, PL4, PL5, INP-an and transformation
- Change concentrations and repeat the experiment of the transformants with PL3 and lldR
08/08/15
- Colony PCR of all Trafos from Friday
- Redo assemblies of INP-AnV and PL(5)
- ON of INP-AnV and PL(5) from assemblies from friday
- Transformation of assemblies
09/08/15
- Redo assemblies of PL(3) and PL(4)
- Trafo of assemblies
- Miniprep ON from Saturday
- Make ON of c89-c98
10/08/15
- Colony PCR of PL(3) and PL(4)
- Make ON cultures of PL(3) and PL(4)
- Send things for sequencing (INP-AnV and PL(5))
- Assemble PL(9)
- Transform assembly of PL(9)
- Lactate calibration curve
- Lactate experiment mammalian cells
- Data evaluation
- Overnight cultures of INP-annexins and c89-c98
- Lactate experiment e coli
11/08/15
- Cryostocks PL3, Pl4, lldr, miniprep, sent for sequencing
- Colony PCR PL9
- Lactate experiment with bacteria twice
- ON of pseva271
- Colony PCR of PL(9), make ON if it worked (didn't work)
- Throw away not confirmed cryos
- Redo PL9 assembly and trafo
- Do new assemblies with just arrived primers
12/08/15
- Miniprep and digest bb
- PL4 and PL3-3 sent for sequencing
- Colony PCR of PL(9), didn't work
- Redo assembly for PL(9) with different backbones and transform
- Assemble and transform lldR-lldP, annexinseva, annexinbb, inpyfp
- TRAIL experiment
- Lactate addition experiment with bacteria twice
13/08/15
- Colony PCR
- Replan mammalian cell experiment: why TRAIL is not working?
14/08/15
- Cryostocks of new cultures
- Colony PCR of PL9
- Miniprep of lldrp, pl9, pl3, pl4 and AnnBBBB and sent some for sequencing
- Miniprep of lldrp, pl9, pl3, pl4 and AnnBBBB and sent some for sequencing
- Preparation of apoptotic cells (TRAIL)
- Co-culture HL-60 and bacteria
- Split cells
15/08/15
- Lactate dose-response experiment with bacteria
- Darmstadt visit
16/08/15
- Lactate dose-response experiment with bacteria
- Darmstadt visit
17/08/15
- Send for sequencing lldrp, pl9 etc
- Good ones PL3 measured at high conc
- Bad ones (65-68) PL(3) measured at same conc
- Microscopy TRAIL
- Miniprep
- Repeat lactate measurement for cells at 6h
- Make overnight cultures of PL(3) versions, top10, lldr, lldplldr
18/08/15
- Bad ones of PL(3) in plate reader at 25mM, 6.3, 3.1, 390uM, 100uM, 10uM
- Make fragments from PL(9)
- Measure lactate from mammalian cells from yesterday (morning before other exp.)
- Make competent cells
- Good ones again with high conc at gain 70
- More minipreps
19/08/15
- Remaining bad ones of PL(3) in plate reader all conc
- Send things for sequencing
- Remake fragment f48 failed
- Pick up trihydrochlorine