further work on pcdf6 cloning, second experiment on larvae locomotion

on Monday, December 7th, 2020 12:33 | by

PCR pCDF6 Primer
-12.5 µl primer reverse/forward
-5 µl dNTPs
-2.5 µl Polymerase
-0.57 µl pCDF6 undigested
-50 µl Q5 buffer
-166.93 µl H2O
Agarose-gel electrophoresis
E.Z.N.A. Gel Extraction

pcr1: 153.3 ng/µl
pcr2: 115.7 ng/µl
pcr3: 174.4 ng/µl

pCDF6 NEBuilder Assembly Reaction
-pcr1: 4.85 ng -> 0.88 µl
-pcr2: 4.24 ng -> 1.5 µl
-pcr3: 4.87 ng -> 1.6 µl
-pCDF6 digested: 100 ng -> 2.8 µl
-H2O: 3.22 µl
-10 µl NEBuilder HiFi DNA Assembly Master Mix/Control
PCR
heat shock transformation of the construct into competent E.coli cells
-> plated on LB0+Amp plates
-> no colonies

control: ELAV-Gal4 x UAS-gRNA (n=23)
experimental: ELAV-Gal4 x UAS-Cas9-gRNA (n=23)


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New experiments on Rover/Sitter larvae

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Testing new tracking software: Kinovea

Barplot of the distance of the Canton-S WT larvae of last week. There is no big difference compared to the tracking with Biotrackr.

Food patch experiments on old Rover/Sitter larvae

ForS and ForR larvae raised on new food. There is a bigger difference between the groups.

Distance tracking of old Rover/Sitter larvae

The larvae were filmed for one minute. Larvae that reached the edge of the petri dish before one minute were discarded.
Video recording was started as soon as the larvae started moving.
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Local FoxP knok out GMR20H5

GMR20H05
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Distance tracking of larvae

on Monday, November 30th, 2020 11:57 | by

Testing of the tracking and R Code

1) Small paper ball to simulate a larva, attached to a black string, and moved 2 cm by hand. 2) Measuring the pixel distance of a known real life distance (1 cm). 3) Plot of x and y coordinates shows trajectory of the object. 4) Bar plot of the real life distance shows that tracking and the R code worked .

Distance tracking of Canton-S wild type larvae

1) Larva on agar plate dyed with activated charcoal 2) Barplot of the distance of the Canton-S WT larvae

Problem finding the video time

Output of the csv file of the BioTracker.
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Experiments on larvae locomotion using control and experimental line

on Thursday, November 26th, 2020 10:55 | by

Experiment on larvae using the control line (ELAV-Gal4 x UAS-gRNA) and experimental line (ELAV-Gal4 x UAS-gRNA-Cas9).
The experiment was significant, with the p-value p=0.0003
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PCR with undigested pCDF6

on Monday, November 23rd, 2020 12:54 | by

primer: reverse/forward each 10 µl
dNTPs: 4 µl
buffer: 40 µl
polymerase: 2 µl
H2O: 133.6 µl
undigested vector: 0.4 µl

PCR of undigested vector
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Further experiments on larval locomotion using Canton-S wild type flies

on Sunday, November 22nd, 2020 10:33 | by

All experiments were done using 1 mm graph paper and a microscope to count the boxes more precisely.

Tracking of the larvae using Ctrax

Agar plate dyed with activated charcoal (2 g charcoal/ 1 l H2O)
Tracking with Ctrax software using the Burridan camera setup.
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Dprime and D1 were verified

on Monday, November 16th, 2020 11:14 | by

Dprime
D1
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Local FoxP knock out

GMR65A06
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Experiments on larval locomotion using Canton-S wild type flies

on Friday, November 13th, 2020 11:07 | by

Time Experiments

One larva with a time exceeding 20 minutes was not considered.
Two larvae with a time exceeding 20 minutes were not considered.

Distance Experiments

Experiment 4: Graph Paper

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