Sarah-Lynn Stratil

GeneSwitch experiment

ELAV-Gal4; TubGal80 results

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)

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

Crosses for conditional knockout

further work on pcdf6 cloning, second experiment on larvae locomotion

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

Experiments on larvae locomotion using control and experimental line

PCR with undigested pCDF6

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

Dprime and D1 were verified

pCDF6 cloning with three inserts

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