FoxP knockout in adult flies

on Monday, December 14th, 2020 12:06 | by

Knockout of FoxP in adult Drosophila, using nsyb Gal4 gen-switch line 80699. Flies were kept for two days on RU486 food.

CD8GFP X nSyb-GS, 2d RU486
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Crosses for conditional knockout

on Monday, December 7th, 2020 1:57 | by

CrossTemp. 
ELAV-Gal4;TubGal80 x UAS-gRNA-Cas918 °C (day 1-x)X
ELAV-Gal4;TubGal80 x UAS-gRNA18 °C (day 1-x)X
ELAV-Gal4;TubGal80 x UAS- Cas918 °C (day 1-x)X
ELAV-Gal4;TubGal80 x UAS-gRNA-Cas930 °C 
ELAV-Gal4;TubGal80 x UAS-gRNA30 °C (day 1-x)X
ELAV-Gal4;TubGal80 x UAS- Cas930 °C 
crosses marked with an X are already in 18°C/30°C rooms.
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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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Local FoxP knok out GMR20H5

on Monday, December 7th, 2020 9:55 | by

GMR20H05
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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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Category: Foxp | No Comments

Local FoxP knock out

on Monday, November 16th, 2020 8:47 | by

GMR65A06
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pCDF6 cloning with three inserts

on Sunday, November 8th, 2020 7:33 | by

pCDF6 digestion with enzyme Bbs1
-5.5µl vector (undigested), conc.: 539.1 ng/µl
-1 µl enzyme (Bbs1)
-5 µl buffer (CutSmart)
-38.5 µl H2O
Agarose-gel electrophoresis
E.Z.N.A. Gel Extraction-DNA Purification from Agarose gel

7Concentration: 38,0 ng/µl
pCDF digested
concentration: 38,0 ng/µl

PCR pCDF6 Primer
-12.5 µl primer reverse/forward
-5 µl dNTPs
-2.5 µl Taq-Polymerase
-0.29 µl pCDF6 digested
-50 µl buffer
-167.5 µl H2O
Agarose-gel electrophoresis
E.Z.N.A. Gel Extraction

Concetration:
primer 1: 170.7 ng/µl
primer 2: 137.4 ng/µl
primer 3: 215.0 ng/µl
primer with 100kb ladder

pCDF6 NEBuilder Assembly Reaction
-pcr1: 4.85 ng -> 1.60 µl
-pcr2: 4.24 ng -> 1.20 µl
-pcr3: 4.87 ng -> 1.20 µl
-pCDF6: 100 ng -> 2.90 µl
-H2O: 3.1 µl
-10 µl NEBuilder HiFi DNA Assembly Master Mix/Control

heat shock transformation
-> plated on LB0+Amp plates
-> no colonies

colony PCR (29.10.20)
-37 µl primer forward/reverse
-37 µl dNTPs
-37 µl Taq Polymerase
-74 µl buffer LSB
-518 µl H2O
Agarose-gel electrophoresis


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Category: Foxp | 1 Comment

Testing of possible Gal4-lines

on Monday, September 28th, 2020 12:11 | by

I finished my cross for the colocalisation of FoxP with the 6 Gal4-lines i ordered.

Two lines show a nice colocalisation and will be tested.

One would show some overlap, but a completly diffrent expression pattern than it should have. So i will try this line again from the stock to exclude a mixup of the line.

The next one dont seem to have any overlap, but also the pattern looks not like it should.

This line seems to have coexpression but the pattern also looks a bit odd, this may be due to a general weak signal and a bad dissection.

The final line seems to have no overlap with the FoxP expression.

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FoxP knock out in adult.

on Monday, September 14th, 2020 1:49 | by

The repetition of the knockout experiment in adult flies indicates that FoxP is not required for learning in adult flies. (Exp = elav tubGal80>Cas9gFoxP)

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