onetaq  (New England Biolabs)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Shared αβ T Cell Receptor Usage in Lungs of Sarcoidosis Patients with Löfgren’s Syndrome

doi: 10.4049/jimmunol.1700570

Figure Lengend Snippet: Pairing of TRAV12-1 + and TRBV2 + CD4 + T cells as determined by emulsion PCR PBMCs were labeled with CFSE, spun, and resuspended in RT-PCR master mix. 300 μl of the oil mixture from the Micellula kit was added on top, and the contents were vortexed for 2.5 minutes. Image was taken at 20× (A). CD4 + T cells were purified from BAL cells or PBMCs from one patient and utilized in emulsion (em) or non-emulsion (non) PCR reactions. A representative 2% agarose gel is shown displaying a DNA ladder (L) and the products obtained after PCR 3 (B). Six DR3 + LS BAL samples were subjected to ePCR, and Vβ usage on TRAV12-1-utilizing BAL CD4 + T cells is shown (C). Vα usage on TRBV2-utilizing BAL CD4 + T cells from six DR3 + LS patients is shown (D). Shown are thirteen αβTCR pairs as determined by ePCR (E). The percentage that the listed TCRα is paired with the listed TCRβ is shown (% α paired with β), as opposed to pairing with other TCRβ in a given patient sample, and vice versa (% β paired with α). Amino acids forming shared or similar CDR3 motifs colored and bolded. TRAV12-1 usage on TRBV2 + cells, TRBV2 usage on TRAV12-1 + cells, TRAV26-1 usage on TRBV20-1 + cells, and TRBV20-1 usage on TRAV26-1 + cells are shown for comparison amongst patient groups (F). Bars represent means ± SD. p values were calculated based on Kruskal-Wallis tests with Dunn’s post-test; * = p

Article Snippet: Cells were centrifuged and resuspended in 50 μl of RT-PCR master mix [1x OneTaq One-Step RT-PCR buffer and enzyme (New England Biolabs, Ipswich, MA), 10 U RNase out (Invitrogen, Carlsbad, CA), 2.5 mg/ml acetylated BSA (Sigma-Aldrich, St. Louis, MO), 400 nM each of the C region primers and Stepout primers, and 40 nM each of the V region primers as described in ].

Techniques: Polymerase Chain Reaction, Labeling, Reverse Transcription Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: The Macerprep: a minimalist kit- and enzyme-free high-yield miniprep utilising alkaline lysis and alkaline hydrolysis principles

doi: 10.1101/2020.08.13.249607

Figure Lengend Snippet: Macerprepped DNA is good template for PCR. 1-3, PCR product by NEB Q5® HiFi polymerase. 4-6, PCR product by NEB Onetaq polymerase. I, 4, Miraprepped DNA. 2, 5, Macerprepped DNA with Solution 3 process. 3, 6, Macerprepped DNA with Solution N3 process. M, marker.

Article Snippet: According to the NEB calculator (NEB, online tool), Tm for NEB Q5 and NEB Onetaq was determined to be 52°C and 61°C.

Techniques: Polymerase Chain Reaction, Marker

Journal: bioRxiv

Article Title: A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing

doi: 10.1101/2020.04.07.029199

Figure Lengend Snippet: qSanger detects SARS-COV-2 RNA when amplified directly from viral particles in transport medium. ( A ) A total of 32 no-template controls, 32 negative control samples (Seracare) and 32 positive samples (Seracare) were assayed. All results were concordant with Seracare and NTC. Three samples were no-calls (undetermined) due to low signal-to-noise ratio in the sequencing results. ( B ) Positive Seracare samples were added to RT-PCR master mix either directly from the VTM or after purification with RNA extraction kit at 125 GCE. The ratio of reference and spike-in intensities were measured by custom data analysis. The mean qSanger ratio was 0.745 (± 0.043 s.e.m., n=8) for direct addition, and 0.97 (± 0.041 s.e.m., n=8) for purified. The coefficient of variation (CV) of positive seracare samples were measured for both Luna and OneTaq polymerase mixes. The CV for Luna direct VTM was 16.4% (n=8), and for Luna purified was 12.1% (n=8). This is consistent with the theoretical counting noise associated with quantifying ~100 molecules.

Article Snippet: qSanger Amplification Reverse transcription and amplification for was performed using OneTaq One-Step RT-PCR Kit from NEB (cat. # E5315S).

Techniques: Amplification, Negative Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Purification, RNA Extraction

Journal: Scientific Reports

Article Title: A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assay

doi: 10.1038/s41598-019-40035-5

Figure Lengend Snippet: Impact evaluation of Master Mix used in singleplex PCR. In graph: Elizyme = 2X EliZyme HS Robust MIX (Elisabeth Pharmacon, Czech Republic); Accustart II = AccuStart II PCR ToughMix (QuantaBio, Massachusetts, USA); Amplitaq = AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Massachusetts, USA); OneTaq = OneTaq Hot Start 2X Master Mix with GC Buffer (New England BioLabs, Massachusetts, USA); Platinum = Platinum Hot Start PCR 2X Master Mix (Invitrogen, California, USA); HotStarTaq = HotStarTaq DNA Polymerase (Qiagen, Germany). YE = Y . enterocolitica; TG = T . gondii ; plus sign in legend = positive sample; minus sign = NTC.

Article Snippet: Several master mixes were tested in the optimization experiments, namely AccuStart II PCR ToughMix (QuantaBio, Massachusetts, USA), AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Massachusetts, USA), OneTaq Hot Start 2X Master Mix with GC Buffer (New England BioLabs, Massachusetts, USA), Platinum Hot Start PCR Master Mix (Invitrogen, California, USA), and HotStarTaq DNA Polymerase (Qiagen, Germany).

Techniques: Polymerase Chain Reaction, Hot Start PCR