λ dna

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

λ DNA is a type of DNA extracted from the bacteriophage lambda. It is a linear, double-stranded DNA molecule that is commonly used as a standard and control in various molecular biology applications, such as DNA sequencing, restriction enzyme analysis, and molecular weight determination.

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12 protocols using λ dna

Spectrophotometric Quantification of DNA

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Estimation of DNA concentration and purity was performed using a spectrophotometer (BioSpectrometer^® basic, Eppendorf AG, Hamburg, Germany) at 260 and 280 nm. For a quantitative determination of DNA, the fluorescence signal was measured after addition of the DNA intercalation dye PicoGreen (Quant-iT™ PicoGreen^® dsDNA Reagent, Thermo Fisher Scientific Inc., Waltham, MA, USA) against a λ-DNA (Thermo Fisher Scientific Inc., Waltham, MA, USA) standard curve using a microtiter plate reader (GENios, TECAN Group Ltd., Männedorf, Switzerland).

Wrage J., Kleyner O., Rohn S, & Kuballa J. (2020). Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR. Foods, 9(3), 332.

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Preparation and Labeling of DNA Samples

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DiYO-labeled DNA was prepared from 500 bp, 10 kbp, and λ-DNA (Thermo Fisher Scientific) and DiYO-1 intercalating dye (AAT Bioquest, Sunnyvale, CA) in a 10 mM KCl buffer, pH 8. A 10:1 base pair-to-dye molar ratio of dsDNA molecules were incubated with DiYO-1 dye for 20 min at 50°C. The 260bp/20kbp SMRTbell template (Pacific Biosciences) was annealed to the primer with 20× stoichiometric ratio in 10× primer buffer. The primer-bound 260 bp/20kbp SMRTbell template (Pacific Biosciences) was incubated with DNA polymerase (Pacific Biosciences P6) with 10× stoichiometric ratio at 30°C for 30 min (proprietary buffer solutions). Samples were then put in 50% glycerol with dithiothreitol and placed at −20°C for storage prior to use.

Farhangdoust F., Cheng F., Liang W., Liu Y, & Wanunu M. (2022). Rapid Identification of DNA Fragments through Direct Sequencing with Electro-Optical Zero-Mode Waveguides. Advanced materials (Deerfield Beach, Fla.), 34(9), e2108479.

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Quantifying cell-free DNA in plasma

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cfDNA in 10 μL platelet-free plasma was measured using an in-house assay, as previously described [14 ]. Briefly, 10 μL of plasma samples from healthy controls or burn patients were added to black opaque Corning 96-well plates in duplicate (Thermo Fisher; Massachusetts, USA) and incubated with 140 μL of SYTOX™ green dye (Thermo Fisher) at a working concentration of 1 μM (diluted in PBS) for 10 min in the dark. For calibration a DNA standard curve (0–1000 ng/mL) of λ-DNA (Thermo Fisher) was included in each assay. Healthy control plasma was added to each plate as a negative control. All samples were run in duplicate. Fluorescence was measured using a BioTek Synergy 2 fluorometric plate reader (NorthStar Scientific Ltd; Potton, UK) with excitation and emission wavelengths set at 485 nm and 528 nm, respectively.

Cato L.D., Bailiff B., Price J., Ermogeneous C., Hazeldine J., Lester W., Lowe G., Wearn C., Bishop J.R., Lord J.M., Moiemen N, & Harrison P. (2021). Heparin resistance in severe thermal injury: a prospective cohort study. Burns & Trauma, 9, tkab032.

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DNA Samples for Biopolymer Translocation

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For the translocated biopolymer, several DNAs have been used: λ DNA (48.5 kbp, ThermoFisher Scientific), λ DNA/HindIII (125 bp to 23,130 bp, average 14.3 kbp, New England Biolabs), T4 GT7 DNA (166 kbp, NIPPON GENE),

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X174 RF II DNA (5.4 kbp, New England Biolabs), pNEB206A linearized (2.7 kbp, New England Biolabs), pCLIP_f-H2B (6.2 kbp, circular DNA, New England Biolabs), pKLAC2 (9.1 kbp, circular DNA, New England Biolabs), and DNA ladder (100 bp to 1,517 bp, average 710 bp, New England Biolabs). The concentration of DNA was kept constant at 4.9 nM (in phosphate groups equivalent) in buffer Tris-KCl (10 mM) and (ethylenedinitrilo)tetraacetic acid (1 mM) at pH 7.6 (25 ^∘C). The DNAs have been fluorescently labeled with YOYO-1 (ThermoFisher Scientific) at 3.0 nM (about one YOYO-1 molecule available per DNA base).

Molcrette B., Chazot-Franguiadakis L., Liénard F., Balassy Z., Freton C., Grangeasse C, & Montel F. (2022). Experimental study of a nanoscale translocation ratchet. Proceedings of the National Academy of Sciences of the United States of America, 119(30), e2202527119.

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Quantifying Circulating Cell-Free DNA

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In order to measure the cfDNA concentration in plasma samples, a fluorometric assay using SYTOX® Green Dye (Life Technologies, Cheshire, UK) was used as described previously [13 (link)]. For each plate, a standard curve was generated using samples of λ-DNA (Fisher Scientific, UK) ranging from 0–1000 ng/ml. To ensure that the assay was reliable between samples and assays, the inter-assay and intra-assay coefficients of variation were calculated; these were 5.3% and 5.1% respectively. 140 μl of SYTOX® Green Dye (Final concentration 1 μM) was added to 10 μl of plasma, and incubated for 10 minutes at room temperature in the dark. All samples were run in duplicate. Fluorescence was then measured using a BioTek Synergy 2 fluorometric plate reader (NorthStar Scientific Ltd, UK). Calibration was set at 485 nm and 528 nm for excitation and emission respectively. The average values of the duplicate wells were then used to derive cfDNA concentrations by interpolating from the standard curve.

Naumann D.N., Hazeldine J., Dinsdale R.J., Bishop J.R., Midwinter M.J., Harrison P., Hutchings S.D, & Lord J.M. (2017). Endotheliopathy is associated with higher levels of cell-free DNA following major trauma: A prospective observational study. PLoS ONE, 12(12), e0189870.

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Patterned Charge-Functionalized Glass Substrates for DNA Immobilization

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Aside from patterned FTO substrates, glass substrates with patterned surface-charge were also fabricated for DNA immobilization. The charge-patterned glass substrates were made by simply using TGA as thiol 2 if CAH was thiol 1, and vice versa (Figure 1). These patterned substrates will be referred to as Glass-CAH-TGA for the first case, and Glass-TGA-CAH in the second case. Fluorescent ATTO 633-NHS dye was used to label the amine moieties (from the surface-bound CAH molecules). Fluorescent images of dye-labeled surface were obtained with a Nikon eclipse Ti inverted fluorescence microscope (with 20× Nikon objective) equipped with an Andor iXon Ultra 897 EMCCD camera. The samples were excited with a 640 nm laser (Dragon Lasers, China).
To demonstrate the patterned charges on the glass surface, we have combed DNA strands on the positively-charged, amine-functionalized regions of Glass-CAH-TGA substrate. This was accomplished by pipetting 100 μL of a 16 pM λ-DNA (Fisher Scientific) in 1×PBS (pH 7.5, Fisher) solution containing 39 nM YOYO-1 (Fisher) onto a patterned glass substrate tilted at an 80° angle. After rinsing the excess DNA solution from the substrate with water, fluorescent images of the combed λ-DNA strands (length: 16 μm) were recorded under 473 nm laser (Dragon Lasers, China) illumination.

Sy Piecco K.W., Vicente J.R., Pyle J.R., Ingram D.C., Kordesch M.E, & Chen J. (2019). Reusable Chemically-Micropatterned Substrates via Sequential Photoinitiated Thiol-Ene Reactions as Template for Perovskite Thin-Film Microarrays. ACS applied electronic materials, 1(11), 2279-2286.

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Quantification of Plasma Cell-Free DNA

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Levels of
cfDNA were measured by fluorometric assay using SYTOX® Green dye (Life Technologies, Warrington, UK). Some 10 μl plasma was incubated with 5 μmol/l SYTOX® Green dye for 10 min and fluorescence was measured using a BioTek® Synergy 2 fluorometric plate reader (NorthStar Scientific, Potton, UK) with excitation and emission set at 485 and 528 nm respectively. For calibration of samples, a λ‐DNA (Fisher Scientific, Loughborough, UK) standard curve was used. The interassay and intra‐assay coefficients of variation were 5·3 and 5·1 per cent respectively.

Dinsdale R.J., Hazeldine J., Al Tarrah K., Hampson P., Devi A., Ermogenous C., Bamford A.L., Bishop J., Watts S., Kirkman E., Dalle Lucca J.J., Midwinter M., Woolley T., Foster M., Lord J.M., Moiemen N, & Harrison P. (2019). Dysregulation of the actin scavenging system and inhibition of DNase activity following severe thermal injury. The British Journal of Surgery, 107(4), 391-401.

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Quantification of cfDNA and MPO-DNA Complexes

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cfDNA was quantified in plasma samples using the Quanti-iT PicoGreen kit (Invitrogen) according to the manufacture’s instructions, and concentrations were calculated based on a standard curve of λ DNA (Invitrogen). MPO-DNA complex levels were quantified by ELISA using a protocol modified from (37 (link)). Briefly, a 96-well flat-bottom plate was coated with anti-human MPO antibody (Bio-Rad) overnight at 4°C and then washed 4 times with PBS-tween buffer before blocking with PBS/4% BSA. The samples were then incubated with patient plasma for 2 hours at room temperature. After 4 washes with PBS-tween, the quantity of DNA bound to captured MPO was quantified using the Quant-iT PicoGreen kit (Invitrogen) as per the manufacturer’s instructions.

Panda R., Castanheira F.V., Schlechte J.M., Surewaard B.G., Shim H.B., Zucoloto A.Z., Slavikova Z., Yipp B.G., Kubes P, & McDonald B. (2022). A functionally distinct neutrophil landscape in severe COVID-19 reveals opportunities for adjunctive therapies. JCI Insight, 7(2), e152291.

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Quantifying Host Cell dsDNA

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Host cell dsDNA was measured with a PicoGreen dsDNA Quantitation Reagent (Invitrogen, Eugene, OR, USA) according to the manufacturer’s instructions. Briefly, samples were transferred to a black flat-bottom 96-well microplate (Thermo Fisher Scientific, Waltham, MA, USA) with λ-DNA (Invitrogen, Eugene, OR, USA) as a standard and mixed with fluorescent dyes (Invitrogen, Eugene, OR, USA). The fluorescence intensity was measured using an Infinite^® 2000 (Tecan, Männedorf, Switzerland) multifunctional microplate reader (~480 nm excitation and ~520 nm emission). All samples were measured in triplicate.

Liu S., Li J., Cheng Q., Duan K., Wang Z., Yan S., Tian S., Wang H., Wu S., Lei X., Yang Y, & Ma N. (2024). A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal. Viruses, 16(5), 768.

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Cucumber Genomic DNA Extraction and Pooling

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Ten seeds per M2 families were sown in small pots inside the glasshouse. Eight cucumber leaf discs (diameter 10 mm) were collected in 96-well plates containing 2 steel beads (4 mm) per well, and tissues were ground using a bead mill. Genomic DNA was isolated using the DNeasy 96 Plant Kit (Qiagen, Hilden, Germany). All genomic DNA was quantified on a 0.8% agarose gel using λ DNA (Invitrogen, Carlsbad, CA, USA) as a concentration reference and normalized to 40 ng/µl. DNA samples were diluted tenfold and pooled eightfold in a 96-well format.

Boualem A., Fleurier S., Troadec C., Audigier P., Kumar A.P., Chatterjee M., Alsadon A.A., Sadder M.T., Wahb-Allah M.A., Al-Doss A.A, & Bendahmane A. (2014). Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics. PLoS ONE, 9(5), e97963.

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