Peqstar 2 pcr machine

Standard molecular biology methods were used, and all constructs were first tested for functionality by transfection into the plasmatocyte-like S2 cell line (Schneider 1972 (link); Woodcock et al. 2015 (link)) (a gift from Frederico Mauri of the Knoblich laboratory at IMBA, Vienna) before injection into flies. Restriction enzymes BSiWI, PstI, and AscI were obtained from New England Biolabs (Frankfurt, Germany); XbaI and EcoRI were from Promega (Mannheim, Germany). PCR amplifications were performed with CloneAmp HiFi PCR Premix from Clontech’s European distributor Takara Bio Europe (Saint-Germaine en Laye, France) using a peqSTAR 2× PCR machine from PEQLAB (Erlangen, Germany). All Infusion cloning was conducted using an Infusion HD Cloning kit obtained from Clontech’s European distributor (see above); relevant oligos were chosen using the Infusion primer tool at the Clontech website http://bioinfo.clontech.com/infusion/convertPcrPrimersInit.do.

Standard molecular biology methods were used, and all constructs were sequenced by the Mycrosynth company (Vienna, Austria) before injecting into flies. The enzymes NotI, T4 Polynucleotide Kinase (PNK), and DpnI were obtained from New England Biolabs (Ipswich, MA, USA). PCR amplifications were performed with GoTaq G2 DNA polymerase (Promega, Madison, WI, USA) using a peqSTAR 2× PCR machine from PEQLAB (Erlangen, Germany). All Infusion cloning was conducted using an Infusion HD Cloning kit (Clontech’s European distributer). The relevant oligo sequences were chosen using the Infusion primer Tool at the Clontech website (Infusion primer design website sInit.do).