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
![7Concentration: 38,0 ng/µl](https://lab.brembs.net/wp/wp-content/uploads/2020/11/pCDF6-digested-943x1024.jpg)
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
![](https://lab.brembs.net/wp/wp-content/uploads/2020/11/primer-pcr-902x1024.jpg)
primer 1: 170.7 ng/µl
primer 2: 137.4 ng/µl
primer 3: 215.0 ng/µl
![](https://lab.brembs.net/wp/wp-content/uploads/2020/11/primer-mit-richtigem-ladder--990x1024.jpg)
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
colony pcr
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.
![](https://lab.brembs.net/wp/wp-content/uploads/2020/09/Overlapping-lines.jpg)
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.
![](https://lab.brembs.net/wp/wp-content/uploads/2020/09/Error-line-2.jpg)
The next one dont seem to have any overlap, but also the pattern looks not like it should.
![](https://lab.brembs.net/wp/wp-content/uploads/2020/09/Error-line-1.jpg)
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.
![](https://lab.brembs.net/wp/wp-content/uploads/2020/09/Fraglich-mit-overlap.jpg)
The final line seems to have no overlap with the FoxP expression.
![](https://lab.brembs.net/wp/wp-content/uploads/2020/09/No-overlap.jpg)
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)
![](https://lab.brembs.net/wp/wp-content/uploads/2020/09/FoxP-cond-knockout.jpg)
Category: flight, Foxp, Memory, operant self-learning | No Comments
PCB x Cas9gFoxP
on Monday, August 31st, 2020 8:47 | by Andreas Ehweiner
Knockout of FoxP in the PCB. Flies are still able to learn.
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/PCB-x-Cas9gFoxP.jpg)
Category: flight, Foxp, operant self-learning | No Comments
August
on Thursday, August 27th, 2020 1:46 | by Ottavia Palazzo
- Halfway in analyzing the Buridan data altogether (not in two batches)
- Maxiprepped plasmid for making FoxP protein at the klinikum
- Writing the thesis a little bit
- bought Fas-II antibody
________________________________________________________
- data 1): FoxP-iB Heterozygous/Homozygous comparison with Stinger-GFP. This time i was cautious with everything: fly all the same age and sex, same larvae density, same number of copies of GFP
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/Picture1-1024x559.png)
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/Picture2-1024x571.png)
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/Picture3-1024x254.png)
I can not detect any difference between homozygous and heterozygous mutants. I also counted cells in IMARIS and I detected no difference in number or distribution.
- data 2): FoxP-iB Heterozygous/Homozygous comparison with CD8-GFP. This time i was cautious with everything: fly all the same age and sex, same larvae density, same number of copies of GFP
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/Picture4-1024x518.png)
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/Picture5-1024x547.png)
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/Picture6-1024x679.png)
I can see some differences but i do not know how to quantify/explain them. Also I am not sure if it is a problem of dissection/mounting. I am not convinced.
- data 3): nc82 staining on homo/hetero FoxP-iB. I do not see differences
![](https://lab.brembs.net/wp/wp-content/uploads/2020/08/Picture0.png)
Problem: I haven’t managed yet to make the FasII antibody work:
I have used a 1:10 concentration of primary antibody for one night and a 1:100 concentration of secondary antibody for 4 hours. I will ask someone
In the picture I have increased the gain a lot just to be sure. I could not see anything
![](https://lab.brembs.net/wp/wp-content/uploads/2020/09/MAX_Project.lif-Series005-1024x1024.jpg)
Category: Foxp | No Comments
Recent experiments
on Monday, June 22nd, 2020 1:52 | by Andreas Ehweiner
Category: flight, Foxp, operant self-learning, set-up test, Uncategorized | No Comments
Pupae TS increased N
on Monday, June 22nd, 2020 10:34 | by Ottavia Palazzo
Category: buridan, Foxp | No Comments
Tdc2-Gal4 + FoxP-LexA in VNC
on Thursday, June 18th, 2020 4:16 | by Ottavia Palazzo
Category: Foxp | No Comments
C380-increased N
on Wednesday, June 17th, 2020 9:49 | by Ottavia Palazzo
Category: buridan, Foxp | No Comments