Molecular biology

3.1.1 Polymerase chain reaction (PCR)

Standard PCRs were conducted in 100 µl total volume with 0.15 mM dNTPs, 4%

(v/v) dimethyl sulfoxide (DMSO), 0.5 µM of each oligonucleotide, 0.001 µg template-DNA and 2.5 u of Pfu-polymerase in its provided buffer in a MyCycler PCR machine.

The following program was used for standard PCR:

94°C 1 minute

94°C 20 seconds

Annealing temperature 30 seconds 30x

72°C 1 min/kb

72°C 4 minutes

10°C ∞

To test a lysogeny broth (LB)-agar-plate for positive clones after ligation and transformation, colony PCRs were performed on single colonies. For this, a colony was picked with a tooth pick which was then put into a PCR tube filled with 10 µl of ddH2O for one minute. Subsequently, the tooth pick was moved to LB-medium (Bertani, 1951) with the appropriate antibiotic (for pGEX-6P-3: ampicillin; pET28a:

kanamycine) and shaken at 37°C, 250 rpm. In parallel, a PCR reaction with the appropriate oligonucleotides was done to detect the desired DNA fragment in the plasmid DNA. Colony PCRs were performed with 20 µl total volume containing 0.125 mM dNTPs, 0.5 µM of each oligonucleotide and 5 u Dream Taq-polymerase in its provided buffer. 1% agarose gels were run to find positive clones, these samples were further shaken at 37°C, 250 rpm over night to prepare plasmids.

• Methods •

The following program was used for colony PCR:

95°C 3 minutes

95°C 20 seconds

Annealing temperature 45 seconds 20x

72°C 1 min/kb

72°C 2 minutes

10°C ∞

In most of the cases, the annealing temperature was 58°C. It was slightly changed to higher or lower values when the first PCR did not show a band on the test agarose gel.

To mutate Otof-C2A (A17D, N68D, S70D and K76D), two mutagenesis steps were performed. First, the N-terminal mutation A17D was inserted by amplifying the whole plasmid with mutated oligonucleotides (16961; 16962). The initial plasmid was digested with DpnI, and the newly synthesized plasmid was electroporated into the E. coli XL1Blue strain. Next, the other three mutations were inserted with help of overlap PCR. Here, two PCRs were done using forward WT oligonucleotide and reverse mutant oligonucleotide (11443 + 17855) for the first PCR, and forward mutant oligonucleotide and reverse WT oligonucleotide (11442 + 17854) for the second PCR. Oligonucleotides were removed afterwards by extracting the PCR products from a 1% agarose gel (3.1.2). The overlapping dsDNA pieces were annealed in an overlap PCR and amplified using the oligonucleotides situated at the ends of the new DNA fragment with the standard PCR protocol. The overlap PCR was conducted in 98 µl total volume with each 10 µl of the two gel eluates, 0.1 mM dNTPs and 2.5 u Pfu-polymerase in its provided buffer.

The following PCR program was used for overlap PCRs:

94°C 5 minutes

37°C 5 minutes 2x

72°C 5 minutes

94°C 1 minute

0.5 µM of each oligonucleotide were added for the standard PCR afterwards.

3.1.2 Restriction enzyme digestion

Prior to restriction enzyme digestions, PCR products were purified with the DNA Clean & Concentrator^TM-5 Kit. Digestions were done in 20-30 µl total volume with 1 µl of each restriction enzyme and 1x appropriate buffer for 1-3 hours at 37°C. After restriction enzyme digestion, the products were loaded on a 1% agarose gel and, after 30 min at 100 V, extracted from the gel with help of Zymoclean^TM Gel DNA Recovery Kit.

For Syt1-C2A-WT, which was kindly provided by Alexander Stein (Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany), NdeI and XhoI were used. For all other constructs, the enzymes indicated in Table 1 were used.

3.1.3 Ligation

Ligations were conducted in 10-20 µl total volume with 0.25-0.5 u/µl T4 DNA Ligase in its supplied buffer at 16°C over night. The molar amount of insert was chosen 4-5 fold higher than that of the vector, according to 1% test agarose gels which were done prior to ligation.

Before transformation, an ethanol precipitation was performed. For this, 3 M sodium acetate (1/10 of the ligation volume) and 100% ethanol (3x the total volume) were added. The mixture was incubated at -20°C for at least 12 hours, then centrifuged for 60 min at 17.000 rcf. The pellet was washed with 70-75% ethanol and centrifuged again for 10 min at 17.000 rcf. The pellet was air dried at 37°C and resuspended in 10 µl ddH2O.

3.1.4 Transformation and sequencing

When cloning plasmid DNA, Escherichia (E.) coli XL1Blue cells were transformed with ligation products (3.1.3) to grow single colonies containing only one specific

• Methods •

DNA plasmid. 50 µl of bacterial suspension were transformed with 1-5 µl of DNA solution in a Micro Pulser^TM electroporator with Gene Pulser® Cuvettes at 1.8 kV.

The cells were diluted in 350 µl LB medium (10 g NaCl, 5 g Bacto Yeast extract, 10 g Bacto Tryptone per one Liter medium) and incubated for 60 minutes at 37°C in an Eppendorf-Thermomixer with 750 rpm. After centrifugation for 1 minute at 17.000 rcf, the cells were resuspended in a small volume (~50 µl) of LB medium and plated on LB agar plates (LB medium with 12 g/L Bacto Agar). The plates contained 25 µg/ml kanamycine when pET28a or pGBK-T7 were used in the transformation, and 100 µg/ml ampicillin/carbenicillin when pGEX-6P-3 or pGAD-T7 were used. The plates were incubated over night at 37°C. Whenever possible, colonies were tested with colony PCR (3.1.1) for the correct insert. Over night cultures were prepared with 5 ml of LB liquid medium containing the appropriate antibiotic and incubated over night at 37° and 250 rpm. The DNA was extracted from the cells with help of the GeneJET^TM Plasmid Minprep Kit.

DNA samples were given to the sequencing service in the Department of Molecular Neurobiology in Max-Planck-Institute for Experimental Medicine, Goettingen, Germany. Sequencing results were evaluated using Chromas Lite (Technelysium Pty Ltd).

Table 7 shows the otoferlin constructs for E. coli expression that have been produced for the project so far.