Isolation of nucleic acids

2.8.1 Mini preparation of plasmid DNA from Escherichia coli (alkaline lysis method) One bacterial colony was inoculated in 3 ml LB medium and the appropriate antibiotics and incubated at 37°C overnight on a shaker. The next day, the cell suspension was centrifuged for 1 min at 13,000 rpm. Afterwards, the cells were resuspended in 200 μl buffer S1, followed by cell lysis for 5 min at RT in 200 μl buffer S2. For neutralization, 200 μl buffer S3 were added and the tube gently inverted. After a 20 min centrifugation step at 13,000 rpm, the supernatant was transferred into a fresh 1.5 ml reaction tube and the DNA was precipitated by adding 400 μl isopropanol. By centrifugation for 30 min at 4°C and 13,000 rpm the DNA was pelleted. This DNA pellet was then washed with 200 μl ethanol (70%) for 5 min at 13,000 rpm. After drying, the pellet was resuspended in 20 μl ddH2O. The isolated DNA was stored at -20°C.

Buffer S1 50 mM Tris-HCl (pH 8) 10 mM EDTA

100 μg/ml RNaseA store at 4°C

Buffer S2 0.2 M NaOH 1% (w/v) SDS store at RT

Buffer S3 3 M KAc pH 5.5 store at RT

2.8.2. Midi Preparation of plasmid DNA from Escherichia coli

This method utilizes the different characteristics of chromosomal and plasmid DNA during the change of the pH value from acidic to basic. The NucleoBond Xtra Midi Kit from Macherey-Nagel was used according to the manufacturers instructions.

For preparation of high DNA amounts, 100 ml LB medium were inoculated with 50 μl bacterial culture including appropriate antibiotics and incubated over night at 37°C on a shaker. The bacteria culture was then transferred to fresh 50 ml falcons and pelleted in a centrifuge (Eppendorf) for 15 min at 4°C (5000 rpm). Meanwhile, the column and the filter were equilibrated with 12 ml buffer EQU. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in 8 ml buffer RES. For cell lysis, further denaturation of DNA and degradation of RNA, 8 ml buffer LYS were added, mixed, and the cell suspension was then incubated for 5 min at RT. Afterwards, 8 ml neutralization buffer NEU were added and the sample was inverted several times to precipitate chromosomal DNA and proteins. The lysate was then loaded onto the column with the filter and washed once with 5 ml buffer EQU. After discarding the filter paper and one washing step with 8 ml buffer WASH each followed. The plasmid DNA stays attached to the exchange matrix during these washing steps. Elution of the plasmid DNA was achieved by adding 5 ml buffer ELU to the column. The plasmid DNA was then

precipitated with 3.5 ml isopropanol, pelleted by centrifugation (30 min at 13,000 rpm at 4°C) and washed with 2 ml 70% ethanol (10 min at 13,000 rpm). Finally, the pellet was dried at RT and solved in 50-100 μl TE-buffer (pH 8.0).

TE-buffer: 10 mM Tris-HCl pH 8.0 1 mM EDTA pH 8.0

2.8.3 Preparation of genomic DNA from Drosophila melanogaster

Thirty flies were frozen for 10 min at -80°C. After adding 200 µl buffer A, the flies were homogenized using a biovortexer. After adding additional 200 µl buffer A, the homogenization was continued until only cuticles remained. The resulting extract was incubated for 30 minutes at 65°C. After addition of 800 μl LiCl / KAc solution (572 µl 6 M LiCl + 228 µl 5 M KAc), the mixture was incubated on ice for 15 minutes and centrifuged at 13.000 rpm for 15 minutes at RT. Then, 1 ml supernatant was transferred into a new reaction tube and 600 µl of isopropanol were added followed by another centrifugation step for 15 min at 13.000 rpm. The pellet was twice washed with 200 µl ice-cold 70%

ethanol and air-dried. Finally, the genomic DNA was dissolved in 150 μl ddH2O or TE-buffer and stored at –20°C for future use.

Buffer A: 100 mM Tris-HCl pH 7.5 100 mM EDTA pH 8.0 100 mM NaCl

0.5 % SDS

2.8.4 Preparation of genomic DNA from single flies

One single fly was shock-frozen in liquid nitrogen. After adding 50 µl squishing buffer and 0.5 µl Proteinase K (200 µg/ml), the fly was homogenized using a biovortexer. The sample was incubated for 30 min at 37°C, then the Proteinase K was inactivated by

another incubation step for 5 min at 95°C. After a final centrifugation step for 5 min at 13.000 rpm, the DNA was either stored at -20°C or 5 µl were directly used for PCR.

Squishing buffer: 10 mM Tris-HCl pH 8.0 1 mM EDTA

25 mM NaCl

2.8.5 Isolation of total RNA from Drosophila melanogaster embryos

Over night embryo collections were used and, if necessary embryos were aged afterwards at 25°C to reach the desired developmental stage. If applicable, embryos were sorted under a fluorescence stereomicroscope (i.e. if balanced over a GFP balancer). For each sample, 25 embryos were collected in a RNAse-free tube. 100 µl NaCl solution (0.9 % in DEPC-H2O) were added and the embryos quickly homogenized with a small pestle. After adding 800 µl TRIzol (QIAGEN) and mixing thoroughly, the sample was incubated at RT for 15 min to allow dissociation. Next, 160 µl chloroform were added, mixed vigorously for 30 sec and incubated for 3 min at RT. After a 20 min centrifugation step at 12.000 x g (4°C) the aqueous phase (upper phase) was cautiously transferred into a fresh RNAse-free tube. To precipitate the RNA, 400 µl isopropanol and 2.2 µl GlycoBlue (Ambion®, Life Technologies AM9515) were added, vortexed for 15 sec and then the sample was incubated at -80°C over night. After 30 min centrifugation at 12.000 x g (4°C), the supernatant was removed and the RNA pellet was washed twice with 75%

ethanol. Finally, the pellet was dried for 5 min at 37°C and dissolved in 20 µl RNAse-free water. If used for transcriptome analysis, 2 µl of the sample were used to check RNA quality and quantity (NanoDrop, BioAnalyzer). The samples were stored at -80°C until further use.