Sarah-Lynn Stratil

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GeneSwitch experiment

on Monday, January 18th, 2021 12:20
larval locomotion
dFoxP knockout in embryonic stage

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ELAV-Gal4; TubGal80 results

on Monday, January 11th, 2021 12:28
ELAV-Gal4; TubGal80: Temperature switch at embryo stage and larvae stage

A,B: KO in embyro stage
C: KO in larvae stage (Experiment 1: in 30 °C for 18 hours, Experiment 2: in 30 °C for 36 hours)

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summary of the results of locomotion experiments on drosophila larvae so far and pcdf6 cloning scheme

on Monday, December 14th, 2020 1:06
t=1 min
method 1: food ring
method 2: one food patch
method 3: edge of petri dish
0.25 cm grid, t=1 min
method 3.1: counting squares
0.1 cm grid under microscope, t= 1min
method 3.2: counting sqaures
Crosses
ELAV-Gal4;TubGal80 x UAS-gRNA
ELAV-Gal4;TubGal80 x UAS-Cas9
ELAV-Gal4;TubGal80 x UAS-Cas9- gRNA
each cross was made 3 times, to put in 18 °C, 25°C and 30°C
A: 25 °C larvae
B: larvae-temperature scheme
30 °C for two days
25 °C until tested

Crosses
ELAV-geneswitch x UAS-gRNA
ELAV-geneswitch x UAS-Cas9
ELAV-geneswitch x UAS-Cas9- gRNA
Crosses for GeneSwitch
pcdf6 cloning scheme

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Crosses for conditional knockout

on Monday, December 7th, 2020 1:57
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

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)


Experiments on larvae locomotion using control and experimental line

on Thursday, November 26th, 2020 10:55
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

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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Dprime and D1 were verified

on Monday, November 16th, 2020 11:14
Dprime
D1
Category: Anatomy | No Comments

pCDF6 cloning with three inserts

on Sunday, November 8th, 2020 7:33

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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