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Plasmid safe dnase

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Plasmid-Safe DNase is an enzyme that selectively digests linear DNA while leaving circular DNA, such as plasmids, intact. It is designed to remove linear DNA contaminants from plasmid preparations without affecting the plasmid DNA itself.

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Lab products found in correlation

Qiaamp dna blood mini kit, by Qiagen (3 mentions) Q5 hot start high fidelity dna polymerase, by New England Biolabs (2 mentions) Gibson assembly, by New England Biolabs (2 mentions) Qiaquick pcr purification kit, by Qiagen (2 mentions) Dna clean up and concentration kit, by Zymo Research (2 mentions) Hydroxyethyl starch viastarch hes, by Fresenius (1 mentions) Irdye800cw nhs, by LI COR (1 mentions) Nonspecific igg, by Abcam (1 mentions) T4 dna ligase buffer, by New England Biolabs (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Herculase 2 dna polymerase, by Agilent Technologies (1 mentions) Abi prism 7900ht system, by Thermo Fisher Scientific (1 mentions) Lightcycler system, by Roche (1 mentions) Tissueruptor, by Qiagen (1 mentions) Qubit 2, by Thermo Fisher Scientific (1 mentions) Plasmid mini kit, by Qiagen (1 mentions) Illustra templiphi kit, by GE Healthcare (1 mentions) Novaseq 6000, by Illumina (1 mentions) Carbenicillin, by Fujifilm (1 mentions) A attachment mix, by Toyobo (1 mentions)

23 protocols using plasmid safe dnase

1

Generating rAAV-based Library from 7-mer Fragment

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The random 7-mer library fragment (inserted between amino acids 588 and 589) was generated by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (NEB; M0493), primers XF and 7×MNN and pCRII-9Cap-xE as a template. A schematic showing the approximate primer binding sites and a table of the primer sequences are given in Supplementary Figure 1 and Supplementary Table 1, respectively. To generate the rAAV-based library, the PCR products containing the library and the XbaI- and AgeI-digested rAAV-ΔCap-in-cis acceptor plasmid were assembled using Gibson Assembly (NEB; E2611). The reaction products were then treated with Plasmid Safe (PS) DNase (Epicentre; E3105K) to digest any unassembled fragments and purified using a QIAquick PCR Purification Kit (Qiagen). This reaction typically yielded over 100 ng of assembled plasmid (as defined by the amount of DNA remaining after the PS DNase digestion step). 100 ng is sufficient to transfect ten 150mm tissue culture dishes at 10 ng/dish. Note, the libraries can also be constructed by ligation or Gibson Assembly and then amplified in E. coli, but bacterial transformation reduces the library diversity. By directly transfecting the assembled reaction products, the library diversity is limited instead by the number of successfully transfected HEK293 producer cells.
Deverman B.E., Pravdo P.L., Simpson B.P., Kumar S.R., Chan K.Y., Banerjee A., Wu W.L., Yang B., Huber N., Pasca S.P, & Gradinaru V. (2016). Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature biotechnology, 34(2), 204-209.
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2

Generating rAAV-based Library from 7-mer Fragment

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The random 7-mer library fragment (inserted between amino acids 588 and 589) was generated by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (NEB; M0493), primers XF and 7×MNN and pCRII-9Cap-xE as a template. A schematic showing the approximate primer binding sites and a table of the primer sequences are given in Supplementary Figure 1 and Supplementary Table 1, respectively. To generate the rAAV-based library, the PCR products containing the library and the XbaI- and AgeI-digested rAAV-ΔCap-in-cis acceptor plasmid were assembled using Gibson Assembly (NEB; E2611). The reaction products were then treated with Plasmid Safe (PS) DNase (Epicentre; E3105K) to digest any unassembled fragments and purified using a QIAquick PCR Purification Kit (Qiagen). This reaction typically yielded over 100 ng of assembled plasmid (as defined by the amount of DNA remaining after the PS DNase digestion step). 100 ng is sufficient to transfect ten 150mm tissue culture dishes at 10 ng/dish. Note, the libraries can also be constructed by ligation or Gibson Assembly and then amplified in E. coli, but bacterial transformation reduces the library diversity. By directly transfecting the assembled reaction products, the library diversity is limited instead by the number of successfully transfected HEK293 producer cells.
Deverman B.E., Pravdo P.L., Simpson B.P., Kumar S.R., Chan K.Y., Banerjee A., Wu W.L., Yang B., Huber N., Pasca S.P, & Gradinaru V. (2016). Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature biotechnology, 34(2), 204-209.
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3

Optimized Trehalose Adjuvant Formulation

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All chemicals were reagent grade. High purity, low endotoxin α,α-trehalose dihydrate was a generous donation of Pfanstiehl (Waukegan, Illinois). Two percent Alhydrogel® (aluminum hydroxide adjuvant) was obtained from Accurate Chemicals and Scientific Corp (Westbury, NY). 3,3′,5,5′-tetramethylbenzidine (Turbo TMB) and peroxidase-conjugated donkey anti-mouse IgG (H + L) was from Thermo Scientific (Rockford, IL). Plasmid-safe DNase was from Epicentre (Madison, WI). Hydroxyethyl starch/Viastarch (HES) was obtained from Fresenius Kabi, Austria, GmbH. IR Dye 800CW NHS was obtained from LI-COR Biosciences, Bad Homburg, Germany.
Garcea R.L., Meinerz N.M., Dong M., Funke H., Ghazvini S, & Randolph T.W. (2020). Single-administration, thermostable human papillomavirus vaccines prepared with atomic layer deposition technology. NPJ Vaccines, 5, 45.
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4

Chromatin Immunoprecipitation of HBV

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Chromatin immune precipitation (ChIP) assays were performed as described29 (link)30 (link). Briefly, 48-hour after transfection with rcccDNA or pTHBV2, Huh-7 cells were fixed in 1% formaldehyde. The cross-linked nuclei were isolated and sonicated in 1% SDS lysis buffer before subjecting to chromatin immunoprecipitation. The reaction was incubated at 4 °C for 14–16 hours after addition of antibodies against H4 (Upstate) and AcH4 (Upstate) and HBcAg (Dako), respectively. Reaction using nonspecific IgG (Abcam) served as negative control. Antibody-DNA-protein complexes were precipitated with protein A/G-conjugated agarose beads and digested with RNase A and proteinase K. The reverse cross-linking was performed at 65 °C for at least 6 hours. DNA was predigested with Plasmid-safe DNase (Epicentre Technologies) before PCR analysis.
Guo X., Chen P., Hou X., Xu W., Wang D., Wang T.Y., Zhang L., Zheng G., Gao Z.L., He C.Y., Zhou B, & Chen Z.Y. (2016). The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely. Scientific Reports, 6, 25552.
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5

Golden Gate Assembly of Plasmid Constructs

Cited 2 times
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The 20 μl volume assembly reaction solution contained 50 ng receptor vector (~3 k bp), 150 ng of each donor vector (~3 k bp), 1.3 μl of each double-stranded oligonucleotides, 1 μl BsaI (10 U, New England BioLabs, R0535), 1 μl T4 DNA Ligase (2000 U, New England BioLabs, M0202) and 2 μl 10 × T4 DNA Ligase buffer (New England BioLabs). The reaction was performed according to Golden Gate protocol, incubated in a PCR instrument for 10 cycles of 5 min at 37 °C and 10 min at 16 °C, then heated to 37 °C for 15 min, 50 °C for 5 min and then 80 °C for 5 min. 1 μl 25 mM ATP and 1 μl Plasmid Safe DNase (10 U, Epicenter) were then added and incubated at 37 °C for 1 h. 5 μl reaction solution was transformed to chemically competent E. coli Trans5α or PXIDF (pSB1s-X). After 1 h incubation at 37 °C with 200 rpm agitation, cells were plated on LB agar containing appropriate antibiotics [26 (link), 28 (link)]. In all, the assembly reaction just needs a few hours to complete.
Zhang S., Zhao X., Tao Y, & Lou C. (2015). A novel approach for metabolic pathway optimization: Oligo-linker mediated assembly (OLMA) method. Journal of Biological Engineering, 9, 23.
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6

Isolation and Quantification of HBV cccDNA

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For nuclei isolation, cells were lysed in fractionation buffer containing 100 mM tris HCl pH 7 and 0.375% NP40 and shook using a vortex for 30 s. Nuclei were pelleted by centrifugation (10 min at 15,000 rpm) and washed in fractionation buffer. Supernatant corresponding to cytoplasmic fractions were kept for RC-DNA quantification. Following centrifugation, DNA was extracted using QIAamp DNA blood mini kit (QIAGEN). For HBV cccDNA quantification, DNA was pre-treated with 10 U of plasmid-safe DNase (EPICENTER) for 1 h at 37 °C, and cccDNA was amplified using cccDNA primers: cccDNA 5′ (5′-GTGCACTTCGCTTCACCTCT-3′) and cccDNA 3′ (5′-AGCTTGGAGGCTTGAACAGT-3′). Samples were normalized using CyclinA2 quantification by qPCR with the following primers: CCNA2 5′ (5′-CCTGCTCAGTTTCCTTTGGT-3′) and CCNA2 3′ (5′-AGACGCCCAGAGATGCAG-3′).
Genera M., Quioc-Salomon B., Nourisson A., Colcombet-Cazenave B., Haouz A., Mechaly A., Matondo M., Duchateau M., König A., Windisch M.P., Neuveut C., Wolff N, & Caillet-Saguy C. (2021). Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein. Scientific Reports, 11, 944.
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7

Oligo Assembly and Purification

Cited 1 time
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Gibson reaction mix was produced as previously reported15 . 4mers were assembled with an equimolar 1mer ratio and an overall DNA input of 120 ng. Reactions were incubated at 50 °C for 30 min with subsequent clean up using Plasmidsafe DNAse (Epicentre). 0.5 μl of final reaction mix was used for PCR amplification with Herculase II DNA Polymerase (Agilent Technologies). PCR amplicons were digested with FastDigest SchI (Life Technologies) directly in PCR buffer for 1 h at 37 °C plus 5 min heat-inactivation at 80 °C. Subsequent 4mer purification utilised Agencourt AMPure XP (Beckman-Coulter) with PEG gradient size selection (final concentration of 7.5% PEG-8000 and 0.9 M NaCl)22 (link).
Gogolok S., Garcia-Diaz C, & Pollard S.M. (2016). STAR: a simple TAL effector assembly reaction using isothermal assembly. Scientific Reports, 6, 33209.
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8

Quantitative Analysis of HBV DNA

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HBV DNA was extracted from plasma and homogenized liver tissue using QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). Plasmatic HBV DNA was quantified by quantitative polymerase chain reaction (qPCR) with a LightCycler system (Roche, Basel, Switzerland) as described by Brezillon et al.21 (link) Intrahepatic HBV DNA was quantified by SYBR Green qPCR on an ABI PRISM 7900HT system (Applied Biosystems, Foster City, CA). For HBV cccDNA, DNA was pre-treated with 10 U plasmid-safe DNase (Epicenter, Madison, WI) for 1 hour at 37°C. HBV-specific primers were: 5′-GTTGCCCGTTTGTCCTCTAATTC-3′ and 5′-GGAGGGATACATAGAGGTTCCTTG-3′ for total HBV DNA; and 5′-GTGCACTTCGCTTCACCTCT-3′ and 5′-AGCTTGGAGGCTTGAACAGT-3′ for cccDNA amplification. Standard curves were generated from an HBV genome-containing plasmid (payw1.2), data was normalized to human cell numbers by qPCR amplification of the human IL8 promoter as described by Rivière et al.23 (link) HBe and HBs antigens were quantified with a clinical test (DiaSorin, Saluggia, Italy).
Dusséaux M., Masse-Ranson G., Darche S., Ahodantin J., Li Y., Fiquet O., Beaumont E., Moreau P., Rivière L., Neuveut C., Soussan P., Roingeard P., Kremsdorf D., Di Santo J.P, & Strick-Marchand H. (2017). Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver. Gastroenterology, 153(6), 1647-1661.e9.
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9

Hepatitis B Virus cccDNA Extraction

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HBV cccDNA was extracted from the liver tissues as previously described with minor modifications [12] (link), [24] (link). Briefly, Liver biopsy tissues were homogenized in a Qiagen Tissue Ruptor after the addition of 200 µl cell lysis buffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.2% NP-40, 0.15 M NaCl] at 4°C, and then centrifuge at 16,000 g for 10 min at 4°C. The pellet was treated with 200 µl nuclear lysis buffer (6% SDS, 0.1 N NaOH), followed by incubation for 30 min at 37°C. The lysate was then neutralized by 100 µl of neutralization buffer [3 M KAc (pH 4.8)] and centrifuged at 16,000 g for 15 min at 4°C. The supernatant was extracted with phenol-chloroform and then chloroform, and the purified precipitate was dissolved in 50 µl TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Potential contaminating host genomic DNA was removed by the Plasmid-Safe DNase treatment (Epicentre Biotechnologies, Madison, WI, USA) and further confirmed by the negativity of GAPDH gene PCR (primers: 5′-ATTCCACCCATGGCAAATTC-3′ and 5′-GGATCTCGCTCCTGGAAGATG-3′) (Fig. S1).
Zhang Y., Mao R., Yan R., Cai D., Zhang Y., Zhu H., Kang Y., Liu H., Wang J., Qin Y., Huang Y., Guo H, & Zhang J. (2014). Transcription of Hepatitis B Virus Covalently Closed Circular DNA Is Regulated by CpG Methylation during Chronic Infection. PLoS ONE, 9(10), e110442.
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10

Isolation and Quantification of HBV cccDNA

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Viral DNAs were precipitated with 35% PEG8000 and isolated from cytoplasmic core particle preparations by proteinase K digestion followed by phenol/chloroform extraction as described previously31 (link). For cccDNA detection, the DNA samples were first pretreated with Plasmid-Safe DNase (Epicenter, Madison, WI). The extracted nuclear DNA was then analysed for cccDNA using TaqMan probe by real-time PCR with the indicated primers (Supplementary Table S1). The precipitated core particle DNA was measured by quantitative PCR or Southern blot. The plasmid pCH9/3091(containing 1.1 copies of HBV genome) served as a template for the standard curve.
Li X., Pan E., Zhu J., Xu L., Chen X., Li J., Liang L., Hu Y., Xia J., Chen J., Chen W., Hu J., Wang K., Tang N, & Huang A. (2018). Cisplatin Enhances Hepatitis B Virus Replication and PGC-1α Expression through Endoplasmic Reticulum Stress. Scientific Reports, 8, 3496.
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