29 dna polymerase

Manufactured by New England Biolabs

Sourced in United States

ϕ29 DNA polymerase is a highly processive and strand-displacing DNA polymerase enzyme that can efficiently synthesize long DNA fragments. It exhibits strong 3' to 5' exonuclease activity, enabling high-fidelity DNA replication.

Automatically generated - may contain errors

Lab products found in correlation

Uracil dna glycosylase, by New England Biolabs (2 mentions) Sep pak c18 cartridge, by Waters Corporation (2 mentions) Oligonucleotide, by Integrated DNA Technologies (2 mentions) Bs poly prep columns, by Bio-Rad (2 mentions) Hinfi, by New England Biolabs (2 mentions) Typhoon 9400 phosphoimager, by GE Healthcare (2 mentions) 29 buffer, by New England Biolabs (2 mentions) Bovine serum albumin (bsa), by New England Biolabs (2 mentions) Spermine, by Merck Group (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Bradford protein stain, by Bio-Rad (1 mentions) Radioactive nucleotides, by PerkinElmer (1 mentions) Unlabeled nucleotides, by GE Healthcare (1 mentions) Dna modification enzymes, by New England Biolabs (1 mentions) Dna oligonucleotide, by Integrated DNA Technologies (1 mentions) T7 dna polymerase, by New England Biolabs (1 mentions) Acrylamide bis acrylamide 19 1 40 solution electrophoresis, by Thermo Fisher Scientific (1 mentions) γ 32p atp 6000 ci mmol, by PerkinElmer (1 mentions) Storm 860, by GE Healthcare (1 mentions) Perfecthyb plus hybridization buffer, by Merck Group (1 mentions)

Spelling variants of this product

DNA polymerase (79 citations)

8 protocols using 29 dna polymerase

Oligonucleotide synthesis and biophysical analyses

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA), [γ-³²P]-ATP (6000 Ci/mmol) was purchased from Perkin-Elmer, uracil DNA glycosylase (UDG) and ϕ29 DNA polymerase were from New England Biolabs (Ipswich, MA, USA), C-18 Sep-Pak cartridges were purchased from Waters (Milford, MA, USA), and BS Poly-prep columns were obtained from BioRad (Hercules, CA, USA). Acrylamide/bis-acrylamide 19:1 (40% solution, electrophoresis grade) was purchased from Fisher Scientific (Waltham, MA, USA), spermine and all other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). A mixture of the four 2’-deoxynucleoside triphosphates (dNTPs) was purchased from Promega (Madison, WI, USA). Iron–EDTA–H₂O₂ footprinting (51 (link)–53 (link)), QTOF-MS (53 (link)–55 (link)), LC–MS/MS (53 (link),55 (link)) and phi-29 (ϕ29) DNA polymerase primer extension reactions (56 (link)) were conducted as described in published procedures, with minor modifications. Detailed experimental protocols for these experiments are provided in the Supplementary Data.

Yang Z., Price N.E., Johnson K.M., Wang Y, & Gates K.S. (2017). Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA. Nucleic Acids Research, 45(11), 6275-6283.

+ Open protocol

+ Expand

T-circle Assay for Detecting ALT Telomeres

Check if the same lab product or an alternative is used in the 5 most similar protocols

T-circle assay was modified from the work of Zellinger et al (2007) (link). 2 μg of genomic DNA was digested with 10 U/μg (each) HinfI and MboI (New England Biolabs) in CutSmart buffer in the presence of 10 μg/ml RNase A overnight at 37°C. 1.5 μg of cut DNA was ethanol-precipitated and resuspended in an annealing buffer (0.2 M Tris, pH 7.5; 0.2 M KCl; and 1 mM EDTA) with 1 μM (CCCTAA)₃ primer containing thiophosphate linkages between the three 3′ terminal nucleotides. The mix was denatured at 96°C for 5 min and cooled down to 25°C for 2 h. DNA was ethanol-precipitated and resuspended in 10 μl of H₂O for the T-circle reaction (10 μl sample combined with 10 μl 0.2 mg/ml BSA, 0.1% Tween, 1 mM each dATP, dGTP, dTTP, dCTP, 1× ϕ29 buffer, and 7.5 U ϕ29 DNA polymerase [New England Biolabs]). Primer extension was carried out at 30°C for 12 h. The ϕ29 DNA polymerase was inactivated by incubation at 65°C for 20 min. The extension products were separated by agarose gel electrophoresis (0.6% agarose at 2 V/cm for 16 h). The gel was then dried at 50°C for 2 h, denatured and hybridized with a specific telomeric probe. U2OS genomic DNA of the ALT cell line was used as a positive control.

Majerska J., Feretzaki M., Glousker G, & Lingner J. (2018). Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1. Life Science Alliance, 1(4), e201800121.

+ Open protocol

+ Expand

Protein Purification and Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols

Radioactive nucleotides were from Perkin Elmer. Unlabeled nucleotides were from GE Healthcare. DNA modification enzymes and ϕ29 DNA polymerase were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies. Protein concentrations were determined using the Bio-Rad Bradford Protein stain and bovine serum albumin as a standard. Buffer A is 20 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA, and 4% glycerol. Buffer B is the same as buffer A except 20 mM Tris-acetate, pH 7.5 was used in place of 20 nM Tris-HCl. Buffer C is 25 mM Tris-Cl pH 7.9, 10% glycerol, 1 mM DTT, 1 mM MgCl₂, 5 mM imidazole, 20 mM KOAc, and 350 mM KCl. Buffer D is 25 mM Tris-OAc pH 7.6, 40 mM K-OAc, 40 mM K glutamate, 2 mM Mg-OAc₂, 1 mM DTT, 20% glycerol, and 0.25 mM EDTA. Stop buffer is 1% SDS, 40 mm EDTA. Buffer H is 20 mM Hepes pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM DTT, 350 mM KCl, 1 mM ATP, and 4 mM MgCl₂.

Georgescu R.E., Schauer G.D., Yao N.Y., Langston L.D., Yurieva O., Zhang D., Finkelstein J, & O'Donnell M.E. (2015). Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation. eLife, 4, e04988.

+ Open protocol

+ Expand

C-circle Assay Protocol for Telomere Length

Check if the same lab product or an alternative is used in the 5 most similar protocols

The method employed for the assay probing for C-circles was slightly modified from that performed in Henson et al.^{19 (link)}. Briefly, genomic DNA was prepared as above. Digested DNA was cleaned up by phenol-chloroform extraction and precipitation. DNA was diluted and measured using a Nanodrop spectrophotometer. Generally, 100 and 50 ng of DNA were used for each sample (10 μl). DNA was combined with 10 μl 0.2 mg/ml BSA (NEB), 0.1% Tween, 1 mM each dATP, dGTP and dTTP, 1X ϕ29 Buffer (NEB) and 7.5 U ϕ29 DNA polymerase (NEB) and incubated at 30 °C for 12 h then 65 °C for 20 min. Reaction products were diluted to 100 μl with 2XSSC and dot-blotted onto a 2XSSC soaked nylon membrane. DNA was UV-cross-linked onto the membrane, and then hybridized at 50°C with end-labeled ³²P-(CCCTAA)₄ oligo probe. Blots were washed, exposed and scanned using a Typhoon 9400 PhosphoImager (Amersham, GE Healthcare) and analyzed using ImageQuant Software.

Rivera T., Haggblom C., Cosconati S, & Karlseder J. (2016). A balance between elongation and trimming regulates telomere stability in stem cells. Nature structural & molecular biology, 24(1), 30-39.

+ Open protocol

+ Expand

C-circle Assay Protocol for Telomere Length

Check if the same lab product or an alternative is used in the 5 most similar protocols

+ Open protocol

+ Expand

C-circle and G-circle Assays for ALT Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols

C-circle assay was performed as described in (50 (link)). Briefly, 300 ng of genomic DNA was digested with HinfI (NEB Inc.) for 1 h at 37°C. Duplicated samples were undigested or digested with 10 units of exonuclease V (Exo V) in the buffer supplemented with 1 mM adenosine triphosphate (ATP), as recommended by manufacturer (NEB Inc), for 1 h at 37°C. For C-circle detection, the ϕ29 DNA polymerase reactions contained the digested genomic DNA as indicated, 0.2 mg/ml BSA, 0.1% Tween-20, 1 mM each of dATP, dGTP and dTTP (or only dATP and dTTP), 1 x ϕ29 buffer and 7.5 U ϕ29 DNA polymerase (NEB Inc.) or no ϕ29 DNA polymerase as control. The reactions were incubated overnight at 30°C, and then the ϕ29 DNA polymerase was inactivated by incubation at 65°C for 20 min. Genomic DNA from U2OS ALT cells and immortalized human fibroblasts were used as positive and negative controls, respectively. The reaction products were run on 0.6% agarose gel in 0.5 × TBE, the gel was dried and hybridized with a 5′ end-labeled (AACCCT)₃ oligonucleotide probe, as described under ‘in-gel hybridization analysis’. G-circle assay was performed in a similar manner, except for using dATP, dCTP and dTTP (or only dATP and dTTP) in the ϕ29 polymerase reaction and 5′ end-labeled (AGGGTT)₃ hybridization probe.

Klebanov-Akopyan O., Mishra A., Glousker G., Tzfati Y, & Shlomai J. (2018). Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection. Nucleic Acids Research, 46(15), 7757-7771.

+ Open protocol

+ Expand

Oligonucleotide Synthesis and Labeling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Uracil DNA glycosylase (UDG), ϕ29 DNA polymerase, and T7 DNA polymerase were from New England Biolabs (Ipswich, MA). A mixture of the four 2′-deoxynucleotide triphosphates (dNTP) was purchased from Promega (Madison, WI). [γ-³²P]-ATP (6000 Ci/mmol) was purchased from Perkin-Elmer. C-18 Sep-Pak cartridges were purchased from Waters (Milford, MA), and BS Poly-prep columns were obtained from BioRad (Hercules, CA). Acrylamide/bis-acrylamide 19:1 (40% Solution/Electrophoresis) was purchased from Fisher Scientific (Waltham, MA), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Yang Z., Price N.E., Johnson K.M, & Gates K.S. (2015). Characterization of interstrand DNA-DNA cross-links derived from abasic sites using bacteriophage ϕ29 DNA polymerase. Biochemistry, 54(27), 4259-4266.

+ Open protocol

+ Expand

Quantification of Telomeric DNA Repeats

Check if the same lab product or an alternative is used in the 5 most similar protocols

C-circle assay was performed as previously described^{28 (link)}. Genomic DNA was digested using AluI and MboI (NEB). 30 ng of digested DNA was combined with 0.2 mg ml⁻¹ BSA, 0.1% Tween, 1 mM each dNTP without dCTP, 1× φ29 Buffer (NEB) and 7.5 U ϕ29 DNA polymerase (NEB). Samples were incubated for 8 h at 30 °C followed by 20 min at 65 °C. Samples were then diluted in 2× SSC buffer and dot-blotted onto an Amersham Hybond-N⁺ nylon membrane (GE). Membrane was ultraviolet crosslinked and then hybridized with ³²P-labelled (CCCTAA)₆ oligonucleotides in PerfectHyb Plus Hybridization Buffer (Sigma) overnight at 37 °C. The next day, the membrane was washed twice in 2× SSC buffer, exposed onto a storage phosphor screen (GE Healthcare) and scanned using STORM 860 with ImageQuant (Molecular Dynamics).

Dilley R.L., Verma P., Cho N.W., Winters H.D., Wondisford A.R, & Greenberg R.A. (2016). Break-induced telomere synthesis underlies alternative telomere maintenance. Nature, 539(7627), 54-58.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!