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Katja Kolar's notebook
From 2009.igem.org
Contents |
Week 1+2 (6/15/09 – 6/26/09)
- bootcamp and team challenge time
- constructs 43/50, 43/55 and OH: miniprepped, test digested, repeated the bad ones
- plated Dictiostelium discoideum aka Dicty on a petri dish with liquid media, grew them
Week 3 (6/29/09 – 7/3/09)
- all GEFs: sequenced the source plasmids, transformed into TG1, miniprepped, test digested, designed and ordered oligos for biobricks
- all actin modulators: transformed into TG1, miniprepped, test digested, designed and ordered oligos for biobricks, PCRed, repeated PCR, ran on gel, purified, BP gatewayed, transformed into DH5α
- DOR fusions with actin binding domains: transformed into TG1, miniprepped, test digested
- learned how to use EZ-TAXIScan, how the CellTracker works
Week 4 (7/6/09 – 7/10/09)
- actin modulators: repeated BP gateway and transformation, miniprepped and test digested ActA short, also ordered new reverse oligos (found a deletion in flankies, that obviously decreases BP yields)
- helped Jackie and Eric with minipreps of GPCRs they transformed, test digested all
- microscopy with wild type HL-60 cells aka HL60's:
- 20 min movies (10 s frame, with 20x objective),
- after 10 min stimulated with fMLP (uniform stimulation, various concentrations - started with 100 nM, then lower/higher),
- used 8-well chambers
- --> optimizing all parameters
- about preparing cells for microscopy:
- coated plating surface with fibronectin
- 2x washed with dPBS
- blocked with RPMI with FBS
- plated cells (5-7 days old)
- 1-2x washed, resuspended in RPMI with FBS
- took movies on microscope
- troubleshooting microscopy:
- bad contrast and focus: movies in 24-well plates, varied the final plating volume, amount of fibronectin
- --> meniscus turns out to be the major problem
- basal movement of HL60's is too high: should I change the plating media? sth. else?
- --> started blocking in mHBSS with 1% BSA (always prepare fresh!!), also washed cells with that + starved the cells before plating – RPMI without FBS for 1h, or RPMI with lower % of FBS over night
- all GEFs, hM4D, Rs1.3, ss-FLAG(-YFP): PCRed, ran on gel, also purified, BP gatewayed and transformed into DH5α the successful ones
Week 5 (7/13/09 – 7/17/09)
- transformations from Friday (and Saturday): colonies only on Rs1.3 and AB parts' plates, miniprepped, test digested (ABs are bad), all others than Rs1.3 rePCRed with different polymerase, ran on gel, successful ones purified, BP gatewayed, transformed into DH5α, miniprepped, test digested (SSFYFP and SSF are OK now)
- all actin modulators and GEFs: PCRed with new reverse primers (just in case..), ran on gel, successful ones purified, BP gatewayed, transformed into DH5α
- B2AR and DOR fusions: test digested some more clones
- microscopy with HL60's:
- switched from small iGEM scopes to the Weiner lab scope (yay! :))
- 24-well movies: meniscus problem solved, but to much fibronectin is used for that
- --> switched to glass coverslips with silicon walls
- observed, that the pancake stage lasts for quite some time – new tot. movie time: 40 min, stimulation after 5 or 10 min
- cells prepared with cca. 1-2h starvation in RPMI with + 0,5% FBS
- made phase movies with
- -wild type cells (unstimulated + stimulated with fMLP)
- -hMD4 stables (Hi and Lo version, unstimulated + stimulated with CNO or fMLP)
Week 6 (7/20/09 – 7/24/09)
- all actin modulators: test digested the souce plasmids
- P-Rex, Tiam, hM2D and hM4D: rePCRed, ran on gel, first one is bad, others purified, BP gatewayed, transformed, miniprepped, test digested
- AarI digested and gel extracted: cAR1 (from Dicty team), SSF, SSFYFP, SOS1, β-Pix, LPDsh, VASP, Tuba, ActAsh, ActAlong, hM4D, Vav
- some of them repeated with additional clones
- Vav done in 2 steps (same size as backbone)
- P-Rex, LPDfull: many troubleshooting PCRs, ran on gel, good ones purified, then either BP gatewayed or DpnI digested, A-tailed and topocloned, transformed. twice.
- AarI ligations (cAR1 with FRB, Rs1.3 with SSFYFP and Vav, ActAlong or ITSN), transformed into TOP10, colony PCRed, ran on gel, miniprepped
- AarI ligations (SSFYFP with hM4D and ActAlong/ActAsh/β-Pix/LPDsh/VASP), transformed into TOP10
- microscopy with HL60's:
- made some bright field movies with wt cells (unstimulated + stimulated with fMLP) - CellTracker doesn't work well with phase
Week 7 (7/27/09 – 7/31/09)
- all Friday's ligations with hM4D: colony PCRed, ran on gel, miniprepped (VASP lig. was bad), sequenced
- AarI digested: hM2D, Vav and Tiam, gel extracted hM2D, 2/3 Tiam clones (Vavs both wrong) ... not OK – repeated PCRs with different DNAPs, ran on gel, Vav and P-Rex are OK
- AarI ligations (SSFYFP with Rs1.3 and Tuba/ActAlong/ActAsh/SOS1/β-Pix/LPDsh/VASP + with hM4D and Tuba/ITSN/SOS1/VASP), transformed into TOP10, colony PCRed, ran on gel, miniprepped good ones, colony PCR2
- designed and ordered primers for all GPCRs and RASSLs
- Dock2 QuickChange mutagenesis, DpnI digested, transformed into TOP10 (failed)
- glycerol stocks: SSF, SSFYFP, cAR1, ITSN, Tuba
- LPDfull: miniprepped, AarI digested, ran on gel, EcoRI (topo constructs...) test digested, ran on gel, repeated digestions, more clones miniprepped, test digested
Week 8 (8/3/09 – 8/7/09)
- Friday's PCRs (last bullet): DpnI digested template DNA, purified, A-tailed, topocloned, transformed, miniprepped, test digested
- Dock2: QC repeated
- GPCR and RASSL parts: PCRed, ran on gel, 2 of 16 failed, others OK, DpnI digested template DNA, purified, A-tailed, topocloned, transformed. did the whole thing twice.
- redesigned and reordered primers for one GPCR
- P-Rex and LPDfull: PCRed with another DNAP, ran on gel, LPDfull then DpnI digested, purified, A-tailed, topocloned, transformed, miniprepped, test digested
- microscopy with hM4D HL60's:
- movies with no stimulation, then with stimulation either with CNO, with fMLP or both; tot. time = 15 min
- used »Iowa's chamber protocol« (in Jackie's notebook)
- troubleshooting of incredibly low yields on colony PCR screenings
- digested and gel extracted more of SSFYFP
- LR gatewayed all AarI ligations that were successful until now, tansformed into DH5α, miniprepped, test digested, ran on gel
Week 13 (9/7/09 – 9/11/09)
Payload part of the project aka micro-oxen:
- prepared different types of beads:
- polystyren, Dyna, silica beads (with or without streptavidin)
- coupled ConA or ConA-biotin to the beads
- two ways to do the experiment:
- coated 8-well chambers with fibronectin, washed, blocked
- prepared HL60's
- attached beads to the cells (incubation at 37ºC, light shaking, optimizing the beads/cell ratio), then
- plated it, washed, resuspended
- took movies (stimulation with fMLP)
- also did the negative control experiments
- or
- coated 8-well chambers with fibronectin, washed, blocked
- prepared HL60's, plated them, washed, resuspended
- started taking the movie - after a while added beads - later washed - then stimulated with fMLP - wait for a while more to see if the cells with attached beads are able to move
