Platinum superfi 2 dna polymerase

cDNA was prepared from ≤1 μg RNA using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) and the Universal Adapter primer (Table S14). cDNA was amplified by PCR using a gene-specific forward primer (Table S14), the Universal Amplification Reverse primer (Table S14), and the proofreading, thermostable Platinum SuperFi II DNA Polymerase (Thermo Fisher Scientific). The resultant blunt-end PCR product was cloned into the Zero Blunt^® TOPO^® vector using the Zero Blunt^® TOPO^® PCR Cloning Kit (Thermo Fisher Scientific). Clones were then sequenced using Sanger DNA Sequencing (GENEWIZ).

The full-length coding sequence of the mouse Vgf gene was obtained by PCR amplification using mouse brain cDNA as the template and Platinum SuperFi II DNA polymerase (Thermo Fisher Scientific) and gene-specific primers. The PCR product was then cloned into the CSII-EF lentiviral expression vector. For lentivirus production, the CSII-EF-Vgf plasmid and lentiviral packaging vectors psPAX and pMD2.G were cotransfected into 293T cells with the use of Lipofectamine 3000 reagent (Thermo Fisher Scientific). At 24 h and 52 h post-transfection, the culture medium containing lentiviral particles was collected, centrifuged at 3000×g for 10 min, and filtered through 0.45 μm filters to remove cellular fragments. Virus precipitation was performed by adding 5 × Lentivirus Precipitation Solution (TransGen Biotech) and incubating for 45 min at 4 °C with agitation. After centrifugation at 7000×g for 45 min, the virus pellets were resuspended in DMEM/F12 and kept in aliquots at − 80 °C.
A total of 1 × 10⁶ TU virus was added to fibrinogen solution, which was subsequently mixed with thrombin solution to form fibrin gels as mentioned above.

Genomic DNA was extracted from 1 ml of an overnight culture of C. vaccini DSM 25150^T as previously described by^{32 (link)}. The putative phlACB cluster (~ 2.8 KB) was amplified from C. vaccini DSM 25150^T genomic DNA using the primers cluster F (ATG AAG AAG GCA GGC ATA GTG AGC TAT GGC AG) and cluster R (ATC TTC CAG CAC GAA CTT GTA GGC GTA TTG CCA) using Invitrogen Platinum SuperFi II DNA polymerase (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The gel purified PCR product was reamplified with new primers containing the restriction sites Nco I (5ʹ end) and Hind III (3ʹ end) for cloning into the pET28b expression vector. Following ligation, three independent expression clones with the phlACB cluster were identified from plasmids that had been transformed into E. coli DH 5α. The correct sequence of the amplified phlACB cluster in each clone was verified by comparison to the genome sequence of C. vaccini CR1 following sequencing of each clone using a MiSeq DNA sequencer. The plasmid clones phlACB.1, phlACB.3 and phlACB.5, as well as empty vector pET28b, were individually transformed into BL21 E. coli.

All primers (Table 1) were designed based on immunoglobulin genes found in the EquCab3.0 reference genome (GCA_002863925.1, SAMN02953672, PRJNA421018). Primer sets EQ-HV and EQ-λ comprise the final high-throughput primer sets designed in this study. EQ-HC primers were used to check IgG isotype, and the EQ-λV-LEADER-17mix was used to fill in missing lambda sequences. EQ-κ primers were used to search for possible kappa light chains, while EQ-HV screening primers were used in the initial setup to look for non-dominant HV-gene representatives in bulk sorted cells.
Thawed single B cell samples were reverse transcribed with SuperScript IV in the presence of fresh RNaseOUT, while immunoglobulin genes were amplified from resulting cDNA using Platinum™ SuperFi II DNA Polymerase and confirmed by size on 48-well E-Gel™ 2% Agarose Gels with SYBR™ Safe DNA Gel Stain, according to the manufacturer recommendations (ThermoFisher SCIENTIFIC, Waltham, MA, USA).

Total RNA was extracted from ROs using the PicoPure RNA Isolation Kit (Arcturus, San Diego, CA, USA). First strand cDNA synthesis was performed using the Superscript IV Vilo Master Mix (ThermoFisher) using 200 ng of total RNA per sample.
QPCR analysis was performed using TaqMan Fast Advanced Master Mix (ThermoFisher) following standard cycling parameters using TaqMan assays to identify genes of interest (Table S3). Primers and probes targeting RPGR^ORF15 were designed using the IDT PrimerQuest Tool (https://eu.idtdna.com/PrimerQuest/Home, accessed on 2 February 2022) to specifically amplify the RPGR^ORF15 transcript only (Integrated DNA Technologies, Coralville, IA, USA; Table S3). The mRNA levels for target genes were normalised to the geometric mean of endogenous reference genes hGAPDH and β-ACTIN and expressed as relative expression versus WT ROs. QPCR statistical analysis was completed on delta delta cycle threshold (Ct) values.
RT-PCR was completed to determine the alternative splicing of RPGR exon 10. Primers were designed in PrimerBLAST (Table S1), focusing on the exons surrounding RPGR exon 10. RT-PCR was completed using standard PCR cycling procedures, using Platinum SuperFi II DNA polymerase (ThermoFisher) and 1 µL cDNA per reaction. RT-PCR reactions were analysed on a 1% agarose gel containing DNA SYBR Safe and imaged on a Biorad ChemiDoc MP (Biorad, Hercules, CA, USA).

HIV-1 subtype A/E was isolated from the plasma of a treatment-naive HIV-1⁺ individual collected at the NHTD as previously described (39 (link)). To construct a full-length infectious molecular HIV-1 clone from the isolated virus, a single HIV-1 clone was established using the SupT1 cell line by limiting dilution of the virus. DNA from the infected cells was then extracted and the entire genome was amplified in two fragments using Platinum SuperFi II DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania). The 5′ fragment extended from the 5′ long terminal repeat (LTR) to the vif region, and the 3′ fragment extended from the vif region to the 3′ LTR. The 5′ fragment was amplified using the forward primer 5′-TGGATGGGCTAGTTTACTCCAAGAAAAGGAAAGAG-3′ and reverse primer 5′-GTCGGTGCTTCCGCTTCTTTCTGCCATAGG-3′, and the 3′ fragment was amplified using the forward primer 5′-CAGGGACAGCAGAGACCCAATTTGGAAAGG-3′ and reverse primer 5′-TGCTAGAGATTTTTACTCAGTCTAGAGTGGTCTGAGGG-3′. The amplified products were then cloned into a pCR-TOPO vector (Thermo Fisher Scientific) and sequenced using an ABI Prism 3130 automated sequencer (Thermo Fisher Scientific). The 5′ HIV-1 fragment was excised by restriction enzyme NotI and shared PflMI sites in the vif region and inserted into the 3′ HIV-1 fragment, thus generating the full-length infectious molecular clone VI-157X4.

All target-specific antisense and sense amiRs (amiR* and amiR, respectively) sequences were designed using the Web MicroRNA Designer, WMD3 (https://www.weigelworld.org/). Each amiR constructs used in this study was cloned individually. The vector backbone was PCR amplified using the Platinum SuperFi II DNA Polymerase and the primers vec_fwd and vec_rev (Supplementary Data 1) (ThermoFisher Scientific), digested with DpnI and gel purified (Macherey&Nagel). The template for the vector backbone amplification was a fully functional and binary amiR expression clone in the pGREEN-based vector backbone vector called pG20_MCS_Hyg (Pratt et al. 2020 (link)). For each amiR construct, primers were designed with the amiR* and amiR being flanked by 5′ and 3′ sequences binding to the template vector (Supplementary Data 1). All amiR fragments were PCR amplified with the respective individual primer pairs using the Phusion polymerase (New England Biolabs) and subsequently gel purified (Macherey & Nagel). The vector backbone and the amiR fragments were assembled using Gibson seamless cloning (New England Biolabs) according to the manufacturer's instructions resulting on a functional binary expression vector. Maps of all vectors are provided in Supplementary Data 2.