Invitrogen turbo dna free kit

To validate expression of phaP1, RT-PCR was conducted for the wild type and phaP1 strains under aerobic and anaerobic conditions using the designed oligonucleotides outlined in Table 1. Cultures were grown to mid-exponential on p-coumarate as shown in Fig. 1, saved with RNALater® solution per manufacture instructions, and stored in −80 °C until further processing. RNA was extracted according to conventional chloroform-phenol extraction, and RNA samples were rid of DNA using the Invitrogen™ TURBO™ DNA-Free Kit (ThermoFisher). The RNA samples were purified using the Monarch® RNA Cleanup Kit (New England BioLabs). All samples were confirmed DNA-free due to a lack of a positive band via PCR with the RNA. Particular care was taken to assess degradation of the RNA samples via bleach gels (Aranda et al., 2012 (link)). Only RNA samples that did not yield a DNA band or show degradation via a bleach gel were converted into cDNA using a High-Capacity cDNA Reverse Transcription Kit per manufacture instructions (ThermoFisher). All cDNA samples were tested via RT-PCR using GoTaq® Green Master Mix (Promega) and the 16S rRNA housekeeping gene (RPA_RNA55) to ensure a positive band. If the housekeeping gene did not reveal a positive band from RT-PCR using a certain cDNA sample, that sample was considered too low of quality for further analysis.

Total RNA of the proband and her mother was extracted, according to the manufacturer's instructions, from peripheral blood collected in PAXgene Tubes, using the PAXgene RNA extraction Kit (all by QIAGEN), and processed with the Invitrogen TURBO DNA-free Kit (Thermo Fisher Scientific). Retrotranscription was obtained by using the Invitrogen SuperScript IV VILO cDNA Synthesis Kit (Thermo Fisher Scientific), and the region of interest was amplified with specific oligonucleotides hybridizing exon 2 and exon 4 (Table 2). Amplicons were separated on a 4% low-melting agarose gel, different-sized fragments were cropped out, and purified with the Invitrogen PureLink Quick Gel Extraction Kit (Thermo Fisher Scientific) and then sequenced. Selective polymerase chain reaction (PCR) for WT transcript amplification was performed using specific forward oligonucleotide, targeting the junction between exon 2 and exon 3, and a reverse oligonucleotide matching the junction between exon 5 and 6 (Table 2). The amplification product was purified with Illustra ExoProStar 1-Step (GE Healthcare) and bidirectionally sequenced. Primers were designed by the Primer3Plus online tool. All the fragments were sequenced using the Applied Biosystems BigDye Terminator v1.1 Cycle Sequencing Kit on an Applied Biosystems 3130xl Genetic Analyzer System (Thermo Fisher Scientific).