Biometra personal thermocycler

Automated polymerase chain reaction (PCR) was performed using the OT-2 liquid handling system and a qTOWER 2.2 (Analytic Jena, Jena, Germany) thermocycler. A microtiter plate holding the PCR reactions was prepared by distribut-15 µL manually prepared PCR master-mix, containing Q5® High-Fidelity 2X Master Mix (New England Biolabs GmbH, Frankfurt am Main, Germany) and plasmid pJC1-lrp-brnF'-eyfp [18] as template, to each well and adding 1 µL of each primer. All reagents were kept cool on ice during the pipetting process. The plate was then covered with foil (SIL-VERseal, Greiner Bio-One International GmbH, Kremsmünster, Austria) and placed into the thermocycler (qTOWER 2.2, Analytic Jena, Jena, Germany). Thermocycling was done according to manufacturer's instructions. Manual PCR was performed using a Biometra personal thermocycler (Analytic Jena, Jena, Germany) thermocycler and the Q5® High-Fidelity 2X Master Mix (New England Biolabs GmbH, Frankfurt am Main, Germany), according to manufacturer's instructions.

Gibson assembly [21] was done by combining 2 µL of PCR product with 8 µL of manually prepared Gibson assembly mastermix, containing 2 ng µL -1 of NcoI-cleaved backbone (pEC-Tmob18-lrp-eyfp), in a microtiter plate using the OT-2 or EVO-200. All reagents were kept cool on ice or on the cooling carrier during the pipetting process. The plate was then covered with foil (SILVERseal, Greiner bio-one, Kremsmünster, Austria) and placed in a qTOWER 2.2 (Analytic Jena, Jena, Germany) thermocycler at 50 °C for 1 hour. Manual Gibson assembly of the pEC-Tmob18-lrp-eyfp backbone was done by combining 1 µL of each PCR product (lrp and eyfp) with 3 µL of EcoRI and BamHI cleaved backbone (pEC-Tmob18) and 5 µL of Gibson assembly mastermix. All reagents were kept cool on ice and placed in a Biometra personal thermocycler (Analytic Jena, Jena, Germany) at 50 °C for 1 hour.