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Gelase

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GELase is a laboratory equipment product designed for the extraction and purification of DNA from agarose gels. It facilitates the isolation of specific DNA fragments for further analysis and applications.

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Lab products found in correlation

Quant it dsdna assay kit, by Thermo Fisher Scientific (8 mentions) Large dna purification kit, by Bio-Rad (4 mentions) Nanodrop 1000, by Thermo Fisher Scientific (4 mentions) Chef genomic dna plug kit, by Bio-Rad (3 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions) Lysis buffer, by Bionano Genomics (2 mentions) Te buffer, by Merck Group (2 mentions) Proteinase k, by Roche (2 mentions) Glycogen, by Roche (1 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) S220 sonicator, by Covaris (1 mentions) Microtube afa fiber pre slit snap cap 6 16 mm, by Covaris (1 mentions) Rbc lysis solution, by Qiagen (1 mentions) Puregene proteinase k, by Qiagen (1 mentions) Millipore membrane filter, by Merck Group (1 mentions) Qubit dsdna assay kit, by Thermo Fisher Scientific (1 mentions) Facsaria 2 sorp flow cytometer and sorter, by BD (1 mentions) Chef mammalian genomic dna plug kit, by Bio-Rad (1 mentions) Chloramphenicol, by Illumina (1 mentions) Copy controltm fosmid library production kit, by Illumina (1 mentions)

19 protocols using gelase

1

High-Molecular-Weight Bacterial DNA Extraction

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Megabase-sized bacterial genomic DNA was extracted with the agarose gel plug method. Embedment in the porous agarose matrix protected the DNA from physical shearing while allowing access to restriction enzymes. The bacterial cell pellets were resuspended and embedded in low-melting agarose to form gel plugs. The plugs were treated with proteinase K and lysozyme for cell lysis. After washing several times in 1× Tris-EDTA buffer, the plugs were melted at 70°C for 2 min and equilibrated at 42°C for 5 min before Gelase (Epicentre, Madison, WI) was added to solubilize the sample. The solubilized DNA obtained was concentrated by drop-dialysis with 1× Tris-EDTA buffer for 2.5 h at room temperature. The high–molecular weight DNA samples were then quantified with a Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific, Walthan, MA). The DNA quality was checked by contour-clamped homogenous electric field gel electrophoresis.
Leung A.K., Liu M.C., Li L., Lai Y.Y., Chu C., Kwok P.Y., Ho P.L., Yip K.Y, & Chan T.F. (2019). OMMA enables population-scale analysis of complex genomic features and phylogenomic relationships from nanochannel-based optical maps. GigaScience, 8(7), giz079.
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2

High-Molecular-Weight DNA Extraction

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The C666-1 cell line was washed with phosphate-buffered saline (PBS) and spun down to a pellet. Next, 106 cells/mL were obtained upon resuspension in PBS, and embedded in 1.5 % low-melting agarose plugs in 0.5 × TBE (Tris-Borate-EDTA) (CHEF Genomic DNA Plug Kit, Bio-Rad). Subsequent handling of the DNA followed BioNano Genomics recommended protocols. The agarose plugs were incubated with proteinase K with lysis buffer at 50 °C overnight. The plugs were washed with a wash buffer to stabilize the DNA in the plugs, and the quality was assessed using pulsed-field gel electrophoresis. A plug was then washed with TE (Tris-EDTA) buffer and melted at 70 °C. After being solubilized with 0.4 U of GELase (Epicentre), the purified DNA was subjected to 2.5 h of drop-dialysis and was shredded by nine strokes of gentle pipetting. The viscous DNA was allowed to equilibrate overnight at room temperature to increase the homogeneity. It was then quantified using a Qubit Broad Range dsDNA Assay Kit (Life Technologies).
Li L., Leung A.K., Kwok T.P., Lai Y.Y., Pang I.K., Chung G.T., Mak A.C., Poon A., Chu C., Li M., Wu J.J., Lam E.T., Cao H., Lin C., Sibert J., Yiu S.M., Xiao M., Lo K.W., Kwok P.Y., Chan T.F, & Yip K.Y. (2017). OMSV enables accurate and comprehensive identification of large structural variations from nanochannel-based single-molecule optical maps. Genome Biology, 18, 230.
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3

Shearing and Purification of Genomic DNA

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Above plugs were washed 4 times for 15 minutes in 15 ml of TE buffer on a horizontal platform mixer at 180rpm at room temperature, transferred to 1.5ml eppendorf tubes, melted at 70°C for 2 minutes and equilibrated for 5 minutes in a water bath at 43°C. The plugs were then digested with 0.4U of GELase (Epicentre) for 45 minutes at 43°C and liberated DNA was cleaned by drop-dialysis (dialysis membranes 0.1µm VCWP04700 Millipore, MA, USA) against 15ml TE buffer for 1 hour. 0.1% of SDS was added to the DNA and treated with 80µg of proteinase K (Ambion) for 15 minutes at 50°C. This DNA was brought to a volume of 130 µl with TE buffer and transferred to a Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6×16mm and sheared on a Covaris S220 sonicator for 4 min at 10% duty cycle, peak incident power 175, 200 cycles per burst in a water bath maintained at 4°C. Sonication under these conditions resulted in DNA fragments with a median shear length of 170bp. At this point, sheared DNA from the same sample in different plugs was combined. DNA was precipitated with 1µl of glycogen (Roche, 20 mg/ml) 0.1 volumes of 3M NaOAc pH5.2 and 2.5 volumes of 100% ethanol in dry ice for 15 minutes, and centrifuged at full speed in a standard microcentrifuge at 4 degrees for 15 minutes. The pellet was washed twice with 70% ethanol and solubilized in 70µl of TE low EDTA (10mM TrisHCl pH 8.0, 0.1mM EDTA).
Canela A., Maman Y., Jung S., Wong N., Callen E., Day A., Kieffer-Kwon K.R., Pekowska A., Zhang H., Rao S.S., Huang S.C., Mckinnon P.J., Aplan P.D., Pommier Y., Aiden E.L., Casellas R, & Nussenzweig A. (2017). Genome Organization Drives Chromosome Fragility. Cell, 170(3), 507-521.e18.
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4

High-Molecular-Weight DNA Extraction from Blood

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High molecular weight DNA was extracted both from fresh (<5 days old) and frozen (– 80 °C) whole blood. DNA extraction was performed following the manufacturer’s guidelines (PlugLysis, Bionano Genomics, USA). RBC lysis solution (Qiagen) was used to lyse red blood cells and pellet white blood cells. The white blood cells were re-suspended in cell suspension buffer (Bio-Rad) and embedded into agarose plugs (CHEF Genomic DNA Plug Kit, Bio-Rad) to lessen fragmentation of long DNA molecules during the overnight lysis at 50 °C using a 16:1 ratio of lysis buffer (Bionano Genomics, USA) and Puregene Proteinase K (Qiagen). The plugs were washed with Tris-EDTA buffer and digested at 43 °C with GELase (Epicentre). Extracted high molecular weight DNA was purified from digested materials/enzymes via drop dialysis using Millipore membrane filters (EMD Millipore, USA) placed on Tris-EDTA buffer. DNA quantifications were carried out using Qubit dsDNA assay kits with a Qubit 3.0 Fluorometer (ThermoFisher Scientific).
Barseghyan H., Tang W., Wang R.T., Almalvez M., Segura E., Bramble M.S., Lipson A., Douine E.D., Lee H., Délot E.C., Nelson S.F, & Vilain E. (2017). Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis. Genome Medicine, 9, 90.
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5

Isolation of High Molecular Weight DNA

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High molecular weight (HMW) DNA was prepared according to Šimková et al. (2003) (link) with modifications. Four batches of 700,000 G1-phase nuclei each were sorted into 660 μl IB buffer in 1.5 ml polystyrene tubes using a FACSAria II SORP flow cytometer and sorter (BD Biosciences, San Jose, CA, United States). One 20 μL agarose miniplug was prepared from each batch of nuclei. The miniplugs were treated by proteinase K (Roche, Basel, Switzerland), washed in wash buffer (10 mM Tris, 50 mM EDTA, pH 8.0) four times, and subsequently five times in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). After the plugs had been melted for 5 min at 70°C and solubilized with GELase (Epicentre, Madison, WI, United States) for 45 min, DNA was purified by drop dialysis against TE buffer (Merck Millipore, Billerica, MA, United States) for 90 min.
Yuan Y., Milec Z., Bayer P.E., Vrána J., Doležel J., Edwards D., Erskine W, & Kaur P. (2018). Large-Scale Structural Variation Detection in Subterranean Clover Subtypes Using Optical Mapping. Frontiers in Plant Science, 9, 971.
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6

High-Quality Genomic DNA Extraction

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Cells from the trio cell line were washed with PBS, resuspended in cell suspension buffer, and embedded in thin low-melting-point agarose layers (CHEF Genomic DNA Plug Kit, Bio-Rad). The thin agarose layers were incubated with lysis buffer and proteinase K for 4 hr at 50°. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to 4 hr of drop-dialysis. It was quantified using Nanodrop 1000 (Thermal Fisher Scientific) and/or a Quant-iTdsDNA Assay Kit (Invitrogen/Molecular Probes), and the quality was assessed using pulsed-field gel electrophoresis.
Mak A.C., Lai Y.Y., Lam E.T., Kwok T.P., Leung A.K., Poon A., Mostovoy Y., Hastie A.R., Stedman W., Anantharaman T., Andrews W., Zhou X., Pang A.W., Dai H., Chu C., Lin C., Wu J.J., Li C.M., Li J.W., Yim A.K., Chan S., Sibert J., Džakula Ž., Cao H., Yiu S.M., Chan T.F., Yip K.Y., Xiao M, & Kwok P.Y. (2015). Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays. Genetics, 202(1), 351-362.
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7

High-Molecular-Weight DNA Purification

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Mammalian cells were embedded in gel plugs and High Molecular Weight DNA was purified as described in a commercial large DNA purification kit (BioRad #170–3592). Plugs were incubated with lysis buffer and proteinase K for four hours at 50°C. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to four hours of drop-dialysis. It was quantified using Quant-iTdsDNA Assay Kit (Life Technology), and the quality was assessed using pulsed-field gel electrophoresis.
Young E., Abid H.Z., Kwok P.Y., Riethman H, & Xiao M. (2020). Comprehensive Analysis of Human Subtelomeres by Whole Genome Mapping. PLoS Genetics, 16(1), e1008347.
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8

High-molecular weight DNA Extraction

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High-molecular weight (HMW) DNA extraction was performed as recommended for the CHEF Mammalian Genomic DNA Plug Kit (BioRad #170-3591). Briefly, cells from the YH or NA12878 cell lines were washed with 2x with PBS and resuspended in cell resuspension buffer, after which 7.5 × 105 cells were embedded in each gel plug. Plugs were incubated with lysis buffer and proteinase K for four hours at 50°C. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to four hours of drop dialysis (Millipore, #VCWP04700) and quantified using Nanodrop 1000 (Thermal Fisher Scientific) and/or the Quant-iT dsDNA Assay Kit (Invitrogen/Molecular Probes).
Cao H., Hastie A.R., Cao D., Lam E.T., Sun Y., Huang H., Liu X., Lin L., Andrews W., Chan S., Huang S., Tong X., Requa M., Anantharaman T., Krogh A., Yang H., Cao H, & Xu X. (2014). Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology. GigaScience, 3, 34.
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9

Genomic DNA Fosmid Library Construction

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Twenty micrograms of highly infective genomic DNA of PRV SC was pipetted using 200 μl tip to shear the DNA. The sheared genomic DNA was end-repaired using the End-Repairing Enzyme Mix to generate 5′ phosphorylated DNA. The DNA fragment was analyzed on a 1% gold agarose gel by pulsed-field gel electrophoresis (PFGE), a Lambda DNA Mono Cut Mix (New England BioLabs, Beijing, United Kingdom) as a size marker. DNA fragments ranging from 33 to 49 kb were excised from the gel, recovered using GELase (Epicentre, Madison, United States) and ligated into the pCC1FOS cloning-ready vector at room temperature for 4 h. The ligation mixture was packaged using MaxPlax Lambda Packaging Extracts and transformed into EPI300-T1R cells. Except genomic DNA, all the materials and protocol as mentioned above were supplied by the Copy ControlTM Fosmid Library Production Kit (Epicentre, Madison, WI, United States). Two hundred clones were picked randomly and cultured overnight in 5 ml of LB liquid medium containing 12.5 μg/ml chloramphenicol and 50 μl of auto-induction solution (Epicentre). The fosmids were extracted using ZR BAC DNA Miniprep Kit (Zymo Research, United States), and the terminal of DNA fragment in the fosmids was sequenced based on pCC1FOS sequencing primers. The whole genomic DNA of PRV SC was assembled according to the terminal sequence of DNA fragment.
Qi H., Wu H., Abid M., Qiu H.J, & Sun Y. (2020). Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing. Frontiers in Microbiology, 11, 1168.
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10

Mammalian High Molecular Weight DNA Purification

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Mammalian cells were embedded in gel plugs and High Molecular Weight DNA was purified as described using a commercial large DNA purification kit (BioRad #170–3592). Plugs were incubated with lysis buffer and proteinase K for four hours at 50 °C. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to 2.5 hours of drop-dialysis. It was quantified using the Quant-iT dsDNA Assay Kit (Life Technology), and the quality was assessed using pulsed-field gel electrophoresis43 (link).
Varapula D., LaBouff E., Raseley K., Uppuluri L., Ehrlich G.D., Noh M, & Xiao M. (2019). A micropatterned substrate for on-surface enzymatic labelling of linearized long DNA molecules. Scientific Reports, 9, 15059.
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