Superscript 3 one step rt pcr

The full-length coding region of KLHL3 (NM_017415.2) was amplified from skeletal muscle total RNA (Agilent) by using RT-PCR (reverse transcription–PCR) according to the manufacturer's protocol (SuperScript® III One Step RT-PCR, Invitrogen). Fragments of KLHL3 comprising residues 290–587 and 296–587 were subsequently PCR-amplified adding flanking BglII and NotI restriction and shuttled directly into the bacterial expression vector pGEX6P-1 downstream of GST and a PreScission Protease cleavage site. Point mutations were introduced using the QuikChange® method (Stratagene) in conjunction with KOD Hot Start DNA polymerase (Novagen). KEAP1 (NM_203500.1) was amplified from EST IMAGE 3163902 and subcloned as a BamHI/NotI insert into a modified pFastbacDualbaculoviral vector containing an N-terminal DAC tag [26 (link)] with a TEV (tobacco etch virus) protease site downstream of the DAC tag. DNA sequencing was performed by The Sequencing Service, College of Life Sciences, University of Dundee, U.K. (www.dnaseq.co.uk).
For structure determination, human KLHL2 (IMAGE clone 4791972; residues 294–593) and human KLHL3 (NM_017415.2; residues 298–587) were cloned into the expression vector pNIC28-Bsa4 (GenBank® accession number EF198106) by ligation-independent cloning. This vector encodes for an N-terminal His₆ tag and rTEV (recombinant TEV) cleavage site.

RT-PCR reactions were performed individually using SuperScript™ III One-Step RT-PCR (Invitrogen) with a combination of multiplex primers designed to amplify highly conserved regions of SARS-CoV-2 and MS2 control genomes. Several target and control primers were tested for efficiency and multiplex compatibility (See Supplementary Tables S3, S4 and S5 for primer sequences and combinations used). A total of four different combinations were used (target A + target B + control C), which will be pooled afterwards in a single library well. Assays were carried out following standard protocols recommended for the kit: 11 µL template RNA, 2 µL SuperScript™ III RT/Platinum™ Taq Mix (Thermo Fisher Scientific, Waltham, MA), 25 µL 2X Reaction Mix (Thermo Fisher Scientific, Waltham, MA), 1 µL of each primer 10 µM (multiplex primer 1 forward, multiplex primer 1 reverse, multiplex primer 2 forward, multiplex primer 2 reverse), 0.5 µL of internal control primer forward 10 µM, 0.5 µL of internal control primer reverse 10 µM and 7 µL nuclease-free water for a final reaction volume of 50 µL. The RT-PCR reactions were incubated using the following cycling conditions: 56 °C for 15 min, followed by one cycle of 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 s, 65 °C for 30 s and 72 °C for 5 s and followed by one cycle of 72 °C for 5 min.

Total RNA from PBMCs was extracted using TRIzol (Invitrogen). The integrity of the RNA was measured with Agilent RNA 6000 Nano. SuperScript™ III One-Step RT-PCR(Invitrogen) was used for reverse transcription and amplification. Quantitative PCR of CSRnc transcripts for IGHM, IGHG1, IGHG3, and AID gene was performed using specific primers and TaqMan probes(IDT). The primers and probes used to quantify the CSRnc transcripts are detailed in Table 1. Amplification of HPRT with PrimeTime® Predesigned qPCR Assays was performed as the reference gene. The fold difference was calculated using 2^ΔΔCT, using resting enriched B cells as calibrator and non-B cells as negative control. HPRT was used as normalizer for every condition.