Platinum taq dna polymerase kit

To confirm absence of RNR expression, strains were grown overnight in LB and diluted to 1:100 in 10 mL fresh media after reaching stationary phase. The cultures were then grown for ~3 hr and total RNA was isolated from mid-log phase cultures using TRIzol Max Bacterial Isolation kit (Thermo Fisher, Catalogue# 16096020). RNA was quantified using a Qubit 4.0 fluorometer. Purified RNA was diluted to 300 ng/mL and treated with TURBO DNaseI (Ambion, Catalogue# AM2238). This DNA-free RNA was then subjected to RT-PCR using the SuperScript III One-Step RT-PCR system with Platinum Taq DNA Polymerase kit (Invitrogen, Catalogue# 12574018) with primers specific to the gene of interest (Supplementary file 1B). Mm-dAK expression was also confirmed using RT-PCR. We observed Mm-dAK expression in our ΔRNR line with and without IPTG induction (Figure 1—figure supplement 2). We therefore elected to perform evolution experiments without IPTG.

For further analysis, all positive samples were subjected to one-step RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit, Invitrogen, Carlsbad, CA, USA. In this procedure, we combined both cDNA synthesis (complementary DNA) and PCR amplification in a single tube and used genome-specific primers for all virus fragments: MBTuni-12 [5-ACGCGTGATCAGCAAAAGCAGG] and MBTuni-13 [5-ACGCGTGATCAGTAGTAGAAACAAGG]. Subsequently, the isolated fragments were cloned into the TOPO vector using the TOPO XL-2 Complete PCR Cloning Kit from Invitrogen and then sequenced with M13 primers using the Sanger method. The Sanger reads were assembled with the SnapGene v.2.3.2 software. For segments longer than the Sanger reads, sequencing was performed in several steps. After sequencing with M13 primers, the primers were synthesized for the further sequencing of the remaining part of the segment.

Total RNA was extracted using RNeasy^® Mini kit (Qiagen) according to the manufacturer's instructions. RT-PCR was performed using SuperScript™ III One-Step RT-PCR System with Platinum^® Taq DNA Polymerase Kit (Invitrogen). The following primers were used: human HBP1, 5′-ATCATCTCCTGTACACATCATAGC-3′(F) and 5′-CATAGAAAGGGTGGTCCAGCTTAC-3′(R); 18S, 5′-GTCTGTGATGCCCTTAGATG-3′(F) and 5′-AGCT TATGACCCGCACTTAC-3′(R). Primer sequences for real-time PCR analysis of oral tumor specimens were: HBP1, 5′-GAACCAATTCAGGCTCACA-3′(F) and 5′-TC AAGACTCAATGCTATCAGTATC-3′(R); FOXO1, 5′-AA GAGCGTGCCCTACTTCAA-3′(F) and 5′-CTGTTGTT GTCCATGGATGC-3′(R). Primer sequences for PCR analysis in ChIP assays were: HBP1-125, 5′-TCTTTCG CCCTCTTATTGA-3′ (F) and 5′-GAACTGCCATTCGG TTCTTC-3′ (R); HBP1-336, F 5′-TTGTCCCAGACAC CAAAACA -3′(F) and 5′-GGATTGGACTATTGCCG AGA-3′(R).

To inactivate virus, samples were treated with TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) and subsequently phase separated by chloroform (Merck, Billerica, MA, USA) treatment and centrifugation. Viral RNA was purified from the aqueous phase using a QIAamp Viral RNA minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted RNA was analyzed by an S-segment-targeting real-time RT-PCR, as previously described (32 (link)). The same primers were included in an in-house version of a SYBR green-based assay using a SuperScript III Platinum SYBR green One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA, USA). The cycling parameters were 20 min at 50°C, 5 min at 95°C, and 45 cycles of 10 s at 95°C, 10 s at 55°C, and 30 s at 68°C, followed by melting curve analysis with wavelength filter F1/F2 performed at the end of the assays. The cycling reactions were performed using a capillary Roche LightCycler 2.0 system. In addition, the primers were included in an endpoint RT-PCR using the SuperScript III One-Step RT-PCR System with a Platinum Taq DNA polymerase kit (Invitrogen, Carlsbad, CA, USA). The cycling conditions were 15 min at 50°C, 2 min at 94°C, 45 cycles of 15 s at 94°C, 15 s at 50°C, and 30 s at 63°C, followed by 5 min at 68°C, using a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA).