phusion hf buffer  (New England Biolabs)

Journal: bioRxiv

Article Title: Powassan Virus LB Neurovirulence and Lethality is Determined by Envelope Protein Domain III Residues

doi: 10.64898/2026.03.26.714546

Figure Lengend Snippet: (A) Genetic homology of POWV strains LB and LI9 with differences indicated by white bars. (B) LB genomes (∼11 kb) were cloned into 5 overlapping fragments and purified Phusion amplified fragments were used develop reverse genetics. (C) POWV-LB fragments and UTR linkers, containing an HDVr, SV40pA, and CMVd2 promoter, were CPER circularized prior to transfection. (D) Immunoperoxidase staining of recLB infected VeroE6 cells 2-4 dpi. (E) Immunoperoxidase staining of WT LB or recLB infected VeroE6 cell foci (MOI, 0.1) 4 dpi.

Article Snippet: DNA fragments 1-5 and linker (0.09 pmol each) were CPER amplified in a 25-μL reaction containing 200 μM of dNTPs, Phusion polymerase GC reaction buffer, and 0.5 μL Phusion polymerase (New England Biolabs).

Techniques: Clone Assay, Purification, Amplification, Transfection, Immunoperoxidase Staining, Infection

Journal: bioRxiv

Article Title: Characterization of variants associated with Cerebral Small Vessel Disease identifies a functional SNV in Versican

doi: 10.64898/2026.03.16.712010

Figure Lengend Snippet: A) Schematic of the MPRA synthetic oligonucleotide including a forward primer (F), 102 bp of flanking sequence before, and 102 bp of flanking sequence after, the variant followed by a miniPromotor (miniP)- GFP construct, a 10 bp barcode and reverse primer (R). The oligo library was cloned into the pMPRAv3:Δluc:ΔxbaI vector and transfected into HEK293 cells. DNA and RNA was isolated separately and sequenced using MiSeq. B) Bioinformatic workflow for the MPRA analysis showing how allele-specific enhancers were identified. C) Fold change in expression for alelle specific enhancers in the indicated genes. Blue bars show significant allele-specific activity.

Article Snippet: To amplify the library, 12 independent PCR reactions were set up containing 1 ng the synthesized MPRA oligos, 10 μl of 5X Phusion HF buffer, 200 μM dNTPs, 1-unit Phusion HF DNA polymerase (New England Biolabs), 0.5uM MPRA oligo-f and MPRA oligo-r (Supp.

Techniques: Sequencing, Variant Assay, Construct, Clone Assay, Plasmid Preparation, Transfection, Isolation, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Characterization of variants associated with Cerebral Small Vessel Disease identifies a functional SNV in Versican

doi: 10.64898/2026.03.16.712010

Figure Lengend Snippet: A) Intergenic region between FOXQ1 and FOXF2 showing location of variants that are significant allele-specific enhancers from the MPRA. B) Table showing the log fold change and adjusted p value for the expression difference between major and minor alleles (allele-specific enhancers).

Article Snippet: To amplify the library, 12 independent PCR reactions were set up containing 1 ng the synthesized MPRA oligos, 10 μl of 5X Phusion HF buffer, 200 μM dNTPs, 1-unit Phusion HF DNA polymerase (New England Biolabs), 0.5uM MPRA oligo-f and MPRA oligo-r (Supp.

Techniques: Expressing