Katja Kolar's notebook

From 2009.igem.org

Revision as of 02:54, 21 October 2009

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 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 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

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 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 gel, successful ones purified, BP gatewayed, transformed into DH5α

• B2AR and DOR phusions: 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, stimulations after 5 or 10 min
  • cells prepared with cca. 1-2h starvation in RPMI with + 0,5% FBS
  • made brightfield (or phase) movies with

-wild type cells (unstimulated + stimulated with fMLP)

-hMD4 stables (Hi and Lo version, unstimulated + stimulated with CNO or fMLP)

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