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Katja Kolar's notebook
From 2009.igem.org
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 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 flakies, 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 reparing 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 turnes 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 gel, also purified, BP gatewayed and transformed into DH5α the successful ones
