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Nusieve gtg

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NuSieve GTG is an agarose gel electrophoresis media designed for the separation and visualization of DNA fragments. It provides consistent performance in separating DNA samples across a wide size range.

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Lab products found in correlation

Vibratome, by Campden Instruments (2 mentions) Hiseq 2000 machine, by Illumina (1 mentions) Wizard sv gel and pcr clean up kit, by Promega (1 mentions) Lsm 510, by Zeiss (1 mentions) β agarase, by New England Biolabs (1 mentions) Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions) Sybr gold, by Thermo Fisher Scientific (1 mentions) Ethidium bromide etbr, by Fujifilm (1 mentions) Ultrapure agarose, by Thermo Fisher Scientific (1 mentions)

11 protocols using nusieve gtg

1

Bile Acid Chemotaxis Preparation

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The chemoattractant (bovine bile) was prepared in chemotaxis buffer (CB; sodium phosphate buffer (50 mM; pH 7.0), disodium ethylenediaminetetraacetic acid (EDTA) (10 µM), and glycerol (0.05% (w/v)) [30 (link)]. To obtain physiology relevant concentrations [24 (link),31 (link)], bile was diluted in CB in a series of 10-fold dilutions from 3% to 0.0003% (w/v). All studies were performed using bovine bile since human and bovine biles have similar compositions, but with different proportions of bile acids [32 (link),33 (link)]. All bile solutions were mixed with 2% (w/v) low-melting-temperature-agarose (NuSieve GTG, Lonza) and were drawn into a 1 μL microcapillary tube in preparation for chemotaxis experiments.
Behroozian S., Sampedro I., Dhodary B., Her S., Yu Q., Stanton B.A, & Hill J.E. (2022). Pseudomonas aeruginosa PAO1 Is Attracted to Bovine Bile in a Novel, Cystic Fibrosis-Derived Bronchial Epithelial Cell Model. Microorganisms, 10(4), 716.
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2

Embryonic Corneal Wound Healing Imaging

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Embryos with wounded corneas were collected at 3, 5, 8, 9, 10 and 11 days post wounding (dpw). Following decapitation, eyes were collected in Ringer’s solution and fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.2. Corneas were dissected from the surrounding scleral tissue, mounted on glass coverslips with 50% glycerol in PBS (v/v) and imaged en face for whole-mount imaging. For cross-section imaging, corneas were embedded in 10% low melting point agarose (NuSieve GTG; Lonza, Rockland, ME, USA) as previously described52 (link). A schematic representation of the wounded embryonic cornea, the tissue orientation, and imaging approaches is illustrated in Fig. 1. Tissue sections of approximately 300 µm thick were cut using a vibratome (Campden Instruments Ltd.) and imaged. The wounded corneas were grouped according to the different phases of the wound healing process; early (3–5 dpw), mid (8–9 dpw) and late (10–11 dpw) healing. At least three wounded and unwounded (control) corneas were analyzed in each group.
Koudouna E., Spurlin J., Babushkina A., Quantock A.J., Jester J.V, & Lwigale P. (2020). Recapitulation of normal collagen architecture in embryonic wounded corneas. Scientific Reports, 10, 13815.
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3

Profiling Small RNA Sequencing Libraries

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We constructed piRNA sequencing libraries from the treated total RNAs using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs) following the manufacturer’s instructions. Briefly, the RNAs were ligated with 3’ and 5’ adaptors and converted to cDNA by reverse transcription. PCRs were then performed using different index primers for different individuals. The PCR conditions were: an initial step at 94°C for 30 sec, 12 cycles at 94°C for 15 sec, 62°C for 12 sec and finally 70°C for 15 sec. The amplified libraries were electrophoresed through a 3% NuSieve GTG (Lonza) 3:1 GenePure LE agarose gel (Bioexpress) and bands ~150 bp were excised. We purified the gel slices using the Wizard SV Gel and PCR Clean-up Kit (Promega). We validated the libraries for quantity and quality on a 2100 Bioanalyzer using a High Sensitivity DNA chip according to the manufacturer’s instructions. The libraries were then sequenced on an Illumina HiSeq 2000 machine using single-end 50 base-pair format. The sequences have been deposited to the NCBI short read archive (SRA) under study number [SRP021475].
Ha H., Song J., Wang S., Kapusta A., Feschotte C., Chen K.C, & Xing J. (2014). A comprehensive analysis of piRNAs from adult human testis and their relationship with genes and mobile elements. BMC Genomics, 15(1), 545.
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4

Second Harmonic Imaging of Corneal Buttons

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The 4 kC buttons evaluated by SHG imaging were obtained immediately post surgery and comprised 3 buttons from 3 different patients that had no previous surgical interventions, and 1 button from a patient that had previously undergone epikeratophakia (Epi-KP) surgery at least 10 years prior to corneal transplant surgery. All samples were prepared following previously established methods and imaged using our standard nonlinear optical high resolution macroscopy (NLO-HRMac) protocol (Jester et al., 2010; Winkler et al., 2013 (link)). Briefly, samples were embedded vertically in low melting point agarose (Nu-Sieve GTG, Lonza Group Ltd., Rockland, ME) and 200 μm tissue sections were collected from the central, cone region of the button using a vibratome (Vibratome 1500; Intracel Ltd., Shepreth, UK). Tissue sections were stored in 4% PFA in PBS until imaged using second harmonic generation microscopy as described previously(Winkler et al., 2013 (link)). Briefly, samples were placed on a glass slide under a laser scanning microscope (Zeiss 510 LSM; Carl Zeiss, Inc., Thornwood, NY) and illuminated with 76 MHz, 150 fs laser pulses from a femtosecond oscillator (Chameleon Titanium Sapphire laser; Coherent, Santa Clara, CA) tuned to 820 nm. The backscattered second harmonic signal (390–430 nm) was collected by the LSM’s meta detector.
Mikula E., Winkler M., Juhasz T., Brown D.J., Shoa G., Tran S., Kenney M.C, & Jester J.V. (2018). Axial mechanical and structural characterization of keratoconus corneas. Experimental eye research, 175, 14-19.
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5

In situ Hybridization of Gdnf in P4 Kidneys

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In situ hybridization was conducted as previously described (Ihermann-Hella et al., 2014 (link)). In brief, an anti-sense RNA probe against Gdnf exons was transcribed and hybridized on thick sections derived from P4 kidneys (10-15 sections/kidney, n=3 kidneys/genotype) embedded in 4% low melting agarose (NuSieve GTG, Lonza). BM Purple was used for colorimetric reaction.
Li H., Kurtzeborn K., Kupari J., Gui Y., Siefker E., Lu B., Mätlik K., Olfat S., Montaño-Rodríguez A.R., Huh S.H., Costantini F., Andressoo J.O, & Kuure S. (2021). Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF. Development (Cambridge, England), 148(10), dev197475.
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6

Genetic Sequence Verification of Transgenic Mice

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Selected regions of Tg4510 and rT2/T2 genomic DNA were amplified by PCR using Herculase II Fusion DNA polymerase (Agilent Technologies). For Tau transgene junctions in Tg4510 DNA, PCR and nested PCR reactions were run (Supplementary Tables 3–5). CaMKIIα-tTA transgene junctions were confirmed using rT2/T2 DNA (Supplementary Tables 6 and 7). Gel bands were isolated from a low melting point agarose gel (NuSieve GTG, Lonza) and DNA was purified following digestion with β-agarase (New England Biolabs). Gel-purified PCR products were sequenced with classical Sanger sequencing at the University of Minnesota Genomics Center, Minneapolis, MN.
Gamache J., Benzow K., Forster C., Kemper L., Hlynialuk C., Furrow E., Ashe K.H, & Koob M.D. (2019). Factors other than hTau overexpression that contribute to tauopathy-like phenotype in rTg4510 mice. Nature Communications, 10, 2479.
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7

Whole Mount in Situ Hybridization

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PFA-fixed E13 urogenital blocks were embedded in 4% low melting agarose (NuSieve GTG, Lonza) and 50 µm thick vibratome sections were cut (HM650, Microm Int.) for whole mount in situ hybridization performed with InSituPro automate (Intavis) according [59] .
Ihermann-Hella A., Lume M., Miinalainen I.J., Pirttiniemi A., Gui Y., Peränen J., Charron J., Saarma M., Costantini F, & Kuure S. (2014). Mitogen-Activated Protein Kinase (MAPK) Pathway Regulates Branching by Remodeling Epithelial Cell Adhesion. PLoS Genetics, 10(3), e1004193.
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8

Whole-embryo Vibratome Sectioning

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Lineage-traced whole or half embryos were fixed in 0.5× MEMFA (44 ) overnight at 4 °C. After washing in PBS, single embryos or halves were transferred into 5 mL of 6% (wt/vol) NuSieve GTG (Lonza) low-melting agarose in PBS in a glass vial (Fisher #03-339-25B) at 60 °C, and immediately embedded in an plastic mold (Electron Microscopy Sciences #62352-07). Samples were oriented head down with forceps under the dissection microscope until the agarose solidified. After incubating for 10 min floating on ice water, the excess agarose was trimmed with a Vibratome blade (Electron Microscope Sciences #71990), the embedded sample was removed from the mold, attached with super glue to a specimen block (Leica #39053747), and serially sectioned with a Vibratome (Lancer Vibratome Series 1000) at 100 µm. Sections were picked up from PBS with a cut pipette tip, placed on clean microscope slides, excess PBS removed, and mounted in antifade reagent (Molecular Probes #P36934). Images were acquired with an LSM880 Zeiss confocal microscope equipped with four lasers.
Moriyama Y, & De Robertis E.M. (2018). Embryonic regeneration by relocalization of the Spemann organizer during twinning in Xenopus. Proceedings of the National Academy of Sciences of the United States of America, 115(21), E4815-E4822.
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9

Corneal Development Tissue Imaging

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All samples were dissected, orientated and processed the same way in respect to the choroid fissure, a fixed anatomic point in the developing optic cup. Embryonic corneas were examined by both en face and cross-section (transverse) tissue imaging approaches. For en bloc tissue imaging (Figure 1), corneas were mounted on the sample holder, epithelial side down, and through-focus image stacks were taken from the epithelial towards the endothelial surface (A). For cross-sectional imaging (B), excised corneas were embedded in 10% low melting point agarose (NuSieve GTG; Lonza, Rockland, ME, USA), and tissue sections of 200–250 μm thick were cut using a vibratome (Campden Instruments Ltd.).
Koudouna E., Mikula E., Brown D.J., Young R.D., Quantock A.J, & Jester J.V. (2018). Cell regulation of collagen fibril macrostructure during corneal morphogenesis. Acta biomaterialia, 79, 96-112.
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10

Genotoxic Assay Protocol with Controls

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Ethyl methanesulfonate (EMS, CAS No. 62–50-0; Wako Pure Chemical Industries, Ltd., Tokyo, Japan) was used as a genotoxic positive control substance [14 ]. Physiological saline (PS) was used as a solvent (Otsuka Pharmaceutical Factory, Inc., Tokyo, Japan). Low melting agarose (LMA, NuSieve GTG) was purchased from Lonza (Basel, Switzerland), and standard melting agarose (SMA, Ultra Pure™ Agarose) was purchased from Thermo Fisher Scientific, Inc. (Massachusetts, USA). SYBRⓇ gold was purchased from Thermo Fisher Scientific, Inc. (Massachusetts, USA). Ethidium bromide (EtBr) was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
Kyoya T., Iwamoto R., Shimanura Y., Terada M, & Masuda S. (2018). The effect of different methods and image analyzers on the results of the in vivo comet assay. Genes and Environment, 40, 4.
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