Trehalose measurement in Würzburg with Dennis Pauls
on Monday, November 19th, 2012 6:58 | by Christine Damrau
Draft of protocol
Material:
0.5 µl capillary with adequate pump, 0.5ml and 1.5ml Eppis, ice, peaked stylus, razor blade, 2 forceps (or scissors)
Procedure:
sting 3 holes in a small eppi (size is important, not to small so that enough hemolymph can go through, not to big so that no fat or other dirt is going through), put into big eppi.
20 adult flies on ice, cut their wings (not sure about the reason)
spear the fly’s thorax with the peaked stylus
store flies in the small eppi with holes
centrifuge the small eppi within the big one (1 min, 5000 rpm)
throw away small eppi, cut big eppi (to access the pellet easier), soak the pellet with a capillary
record the amount of soaked hemolymph with a ruler (e.g. by photographing under disscetion scope)
pump the hemolymph out of the capillary into a fresh small eppi and freeze it.
Remarks:
Everything has to be done on ice, cooling chain should as poosible not be interrupted
(5000 rpm can be differ with different centrifuges…)
Category: Biogenic Amines, trehalose measurement | No Comments
First flight tests, Trehalose again…
on Thursday, November 1st, 2012 7:33 | by Christine Damrau
Chrisi’s day…
1 Collected virgins for our ill post-doc, made some crosses:
d42-GAL4 males x tubGAL80-UAS-PKCi and x CS
d42-chaGAL80 males x tubGAL80-UAS-PKCi and x CS
2 Buridan: Prepared colored food (10mg/ml, 2.5ml total in the very little vials, ~ 5 flies in each vial). Denise tested in the afternoon females in the Buridan. Data (4n now) will be shown in the lab meeting.
3 Imaging Made a new cross for the Imaging-project:
TDC2-GAL4 males x UAS-GCaMP3(2)
replaced Neloy’s flies by flies from stock
Florian’s larval preparation (good!) showed that GCaMP-expression is much less than GFP-expression. They hope for the homozygous line.
4 Flight: Performed flight experiments with the tbh,TDC,UAStbh flies. Raw data, see below. Thinking about analysis (probably tomorrow)
Protocol: glued the day before, sugar in wheel chambers, little EVIAN-water.
5s filter on the legs before starting, aspirated, stopped when no response 3 times in a row, stopped time when 30min, stopped after 15 aspiration trials
| date | fly | genotype | number_aspiration | time_to_first_stop | duration_of_flights | remarks |
| 01.11.2012 | 1 | 12 | 4.56 | 4.56,0.22,0.30,0.2 | ||
| 01.11.2012 | 2 | 2 | 49.15 | 49.15,14.42 | ||
| 01.11.2012 | 3 | 16 | 0.03 | 0.03,0.15,3.06,1.56,0.3,0.14,4.58,0.22 | ||
| 01.11.2012 | 4 | 3 | 0 | 0 | ||
| 01.11.2012 | 5 | 40 | 0.05 | 0.05,0.01,0.02,0.01,0.01,0.02,0.02,0.01,0.02,0.03,0.02,0.02,0.01,0.01,0.01,0.01,0.01,0.04,0.09,0.01,0.02,0.01,0.11,0.01,0.01,0.01,0.01,0.01,0.02,0.01,0.03 | ||
| 01.11.2012 | 6 | 3 | 0 | |||
| 01.11.2012 | 7 | 13 | 3.01 | 0.01,0.01,0.01,0.01,0.01,0.02,0.01 | ||
| 01.11.2012 | 8 | 15 | 0.01 | 0.01,0.04,0.03,0.02,1.56,0.15,0.02,1.46,3.26,1.15,0.46,6.45,2.56,0.06,12.59 | ||
| 01.11.2012 | 9 | 15 | 0.01 | 0.01,0.01,0.28,0.1,1.5,1.29,0.40,0.29,1.34,0.54,0.22,0.37,0.31,0.54 | ||
| 01.11.2012 | 10 | 9 | 0.01 | 0.01,0.01,0.01,0.01,0.01 | ||
| 01.11.2012 | 11 | 12 | 0.01 | 0.01,0.01,0.01,0.01,0.01,0.01 | ||
| 01.11.2012 | 12 | 3 | 0 | |||
| 01.11.2012 | 13 | 4 | 0.06 | 0.06,0.5,0.06,38.2 | ||
| 01.11.2012 | 14 | 3 | 0.19 | 0.19,0.33,0.31,30 | ||
| 01.11.2012 | 15 | 15 | 3.38 | 3.38,2.43,0.47,0.36,4.06,0.39,1.25,0.35,0.52,1.07,0.22,0.23,0.27 | ||
| 01.11.2012 | 16 | 3 | 0 | |||
| 01.11.2012 | 17 | 3 | 7.16 | 7.16,23.27,30 | ||
| 01.11.2012 | 18 | 3 | 0 | |||
| 01.11.2012 | 19 | 6 | 1.33 | 1.33,14.57,9.17,30 | ||
| 01.11.2012 | 20 | 1 | 18.20 | stopped the expriment | ||
| 01.11.2012 | 21 | 3 | 2.50 | 2.50,2.18 | stopped the expriment |
5 Trehalose: Found time to try out Gérard’s idea (no pigment in the probes to not disturb the NanoDrop measurement). 4 groups: 10 flies with or without heads, 50 flies with or without heads. Hilde showed me how to remove heads with liquid nitrogen and vortex. The other flies (with heads) were frozen at -20° as usual. Trehalase incubation over night. Tomorrow glucose oxidase and measurement
Category: Biogenic Amines, buridan, flight, trehalose measurement | No Comments