FoxP knockout in adult flies
on Monday, December 14th, 2020 12:06 | by Andreas Ehweiner
Knockout of FoxP in adult Drosophila, using nsyb Gal4 gen-switch line 80699. Flies were kept for two days on RU486 food.
Category: flight, Foxp, Memory, Operant learning, operant self-learning, Uncategorized | No Comments
Crosses for conditional knockout
on Monday, December 7th, 2020 1:57 | by Sarah-Lynn Stratil
Cross | Temp. | |
ELAV-Gal4;TubGal80 x UAS-gRNA-Cas9 | 18 °C (day 1-x) | X |
ELAV-Gal4;TubGal80 x UAS-gRNA | 18 °C (day 1-x) | X |
ELAV-Gal4;TubGal80 x UAS- Cas9 | 18 °C (day 1-x) | X |
ELAV-Gal4;TubGal80 x UAS-gRNA-Cas9 | 30 °C | |
ELAV-Gal4;TubGal80 x UAS-gRNA | 30 °C (day 1-x) | X |
ELAV-Gal4;TubGal80 x UAS- Cas9 | 30 °C |
Category: Foxp | No Comments
further work on pcdf6 cloning, second experiment on larvae locomotion
on Monday, December 7th, 2020 12:33 | by Sarah-Lynn Stratil
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
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
Category: Foxp, Uncategorized | No Comments
Local FoxP knok out GMR20H5
on Monday, December 7th, 2020 9:55 | by Andreas Ehweiner
Category: flight, Foxp, Operant learning, operant self-learning | No Comments
Experiments on larvae locomotion using control and experimental line
on Thursday, November 26th, 2020 10:55 | by Sarah-Lynn Stratil
Category: Foxp | No Comments
PCR with undigested pCDF6
on Monday, November 23rd, 2020 12:54 | by Sarah-Lynn Stratil
primer: reverse/forward each 10 µl
dNTPs: 4 µl
buffer: 40 µl
polymerase: 2 µl
H2O: 133.6 µl
undigested vector: 0.4 µl
Category: Foxp | No Comments
Local FoxP knock out
on Monday, November 16th, 2020 8:47 | by Andreas Ehweiner
Category: flight, Foxp, Operant learning, operant self-learning, Uncategorized | No Comments
pCDF6 cloning with three inserts
on Sunday, November 8th, 2020 7:33 | by Sarah-Lynn Stratil
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
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
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
Testing of possible Gal4-lines
on Monday, September 28th, 2020 12:11 | by Andreas Ehweiner
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.
Category: Anatomy, crosses, Foxp | No Comments
FoxP knock out in adult.
on Monday, September 14th, 2020 1:49 | by Andreas Ehweiner
The repetition of the knockout experiment in adult flies indicates that FoxP is not required for learning in adult flies. (Exp = elav tubGal80>Cas9gFoxP)
Category: flight, Foxp, Memory, operant self-learning | No Comments