Molecular Biology Methods

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T7KozakStartfor GGATCCTAATACGACTCACTATAGGGA

ACAGCCACCATG This work

FLAG-EFTUD2 for CTACAAGGACGACGATGACAAGATGGA

TACCGACTTATATGATG This work

HA-EFTUD2 rev GTAGTCTGGGACGTCGTATGGGTACAT

GGGGTAATTGAGCACAAC This work

HA-EFTUD2 for ATACGACGTCCCAGACTACGCTATGGA

TACCGACTTATATGATG This work

FLAG-EFTUD2 rev GTCATCGTCGTCCTTGTAGTCCATGGGG

TAATTGAGCACAAC This work

Spacer-NdeI-FLAG for GAACAGCATATGGACTACAAGGACGAC

GATGAC This work

Spacer-NheI-Stop-HA rev GAACAGGCTAGCTTAAGCGTAGTCTGG

GACGTCGTAT This work

Spacer-NdeI-HA for GAACAGCATATGTACCCATACGACGTC

CCAGACTAC This work

Spacer-NheI-Stop-FLAG rev GAACAGGCTAGCTTACTTGTCATCGTCG

TCCTTGTAG This work

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Transformation of Chemically Competent Cells 5.2.2

Transformation of the chemically competent cells was performed by the following procedure: A 50 µl aliquot of DH5α cells was thawed on ice for 10 min. Then 1 µl of plasmid DNA with a concentration of 15 ng/µl (in H₂O) was added. After 30 min incubation on ice, the DH5α cells were heat-shocked at 42°C for 45 sec. After 2 min incubation on ice, 800 µl LB medium was added and the cells were incubated for 1 h at 37°C under shaking at 600 rpm. The cells were then pelleted by centrifugation at 16100 g for 15 sec. 650 µl of the supernatant were removed, the pellet was resuspended in the remaining supernatant and distributed on an LB agar plate containing the respective antibiotic. The plate was incubated at 37°C for 16 h.

Preparation of Plasmid DNA 5.2.3

To amplify plasmid DNA, a single colony was used to inoculate 3 ml LB medium containing 100 µg/ml ampicillin or 10 µg/ml kanamycin, or 25 µg/ml chloramphenicol depending on the resistance conferred by the respective plasmid.

For a Miniprep, the cells were grown for 16 h at 37°C and 220 rpm shaking. The Minipreps were done using the Wizard® Plus SV Minipreps DNA Purification System from Promega according to the manufacturer’s instructions, except that addition of Alkaline Protease Solution, which was interfering with subsequent in vitro translation reactions, was omitted. Elution from the Spin Columns was generally done with 50 µl H₂O.

For a Midiprep, the cells were incubated for 8 h at 37°C and 220 rpm shaking after inoculation of 3 ml LB-ampicillin. Then 100 µl were diluted into 50 ml LB-ampicillin and incubated for 16 h at 37°C and 220 rpm shaking. The Midiprep was done using the PureYield™ Plasmid Midiprep System from Promega according to the manufacturer’s instructions. Elution from the PureYield™ Binding Column was generally done with 550 µl H2O. The DNA concentration was determined by absorbance at 260 nm using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Plasmid DNA was stored at -20°C.

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Gel Electrophoresis of DNA 5.2.4

Gel electrophoresis of DNA was generally done for 20 min at 90 V using 1% agarose LE gels in TAE buffer (Table 3). For visualization of DNA, SYBR® Safe was diluted 1:10000 into the gel mixture. As a molecular weight marker, GeneRuler™ 1 kb DNA Ladder from Fermentas was used. Visualization was done using a UV-transilluminator system from MVG-Biotech and the BioCaptMW software. When DNA fragments were to cut from the agarose gel for subsequent applications, the Safe Imager™ 2.0 Blue Light Transilluminator from Invitrogen was used.

Purification of DNA from agarose gel slices was done using the Wizard® SV Gel and PCR Clean-Up System from Promega according to the manufacturer’s instructions.

Polymerase Chain Reaction 5.2.5

Generally, PCR reactions were performed in a volume of 50 µl and contained 15 ng template DNA. The final concentration of each primer was 400 nM and the final concentration of each dNTP was 200 µM. As polymerase, generally Herculase Enhanced DNA Polymerase from Stratagene was used at a final concentration of 0.1 U/µl. The PCR reactions were done in a T3 Thermocycler from Biometra. An initial denaturation step was included in all PCR conditions for 3 min at 95°C. Within each cycle, the denaturation step was generally at 95°C for 30 sec, the annealing temperature was dependent on the respective primers and was generally between 50-60°C for 30 sec, and the elongation step was at 72°C for 1 min per kb of expected PCR product.

Generally, each cycle was repeated 30-35 times, followed by a final elongation step at 72°C that was approximately 1.5 times as long as the elongation step within the cycle. For site directed mutagenesis, the cycle was typically repeated only 15 times. PCR purification was done using the Wizard® SV Gel and PCR Clean-Up System from Promega according to the manufacturer’s instructions.

Restriction Digest 5.2.6

All restriction enzymes were purchased from New England Biolabs. Restriction digests were done in a final volume of 10 µl containing typically 1-2 µg DNA in the buffer best suited for the

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respective restriction enzymes (NEBuffer 1, 2, 3 or 4, New England Biolabs). When suggested for the respective restriction enzymes, the reactions contained additionally 100 µg/ml BSA.

Normally, 0.5 µl of each restriction enzyme were used. The reactions were incubated at 37°C for 90 min. When the respective fragment was to be used as the vector in a subsequent ligation, the reaction was incubated for another 30 min at 37°C after addition of 5 units of Calf Intestinal Alkaline Phosphatase (New England Biolabs) to prevent vector re-ligation.

DNA Ligation 5.2.7

Ligation was done after purification of the respective fragments from agarose gels. The total volume of a ligation reaction was 10 µl and the ligation was done in T4 DNA Ligase Reaction Buffer (New England Biolabs). The molar insert:vector ratio was typically 3-5 and the total DNA mass in the reaction was normally 100-150 ng. Generally, a ligation reaction contained 200 cohesive end units T4 DNA Ligase, purchased either from New England Biolabs or from the core facility of MPIB. The ligation reactions were incubated for 40 min at room temperature followed by incubation for 40 min at 4°C. Prior to transformation of chemically competent DH5α, the T4 DNA ligase was inactivated by incubation at 60°C for 10 min. Typically, 2.5 µl of a ligation reaction were used for transformation of 50 µl DH5α.

The DNA sequence of all constructs was verified by DNA sequencing in the Core Facility of MPIB.