The C-circle assay was performed as described previously (40 ). Briefly, Hinf1/Rsa1/Alu1-digested genomic DNAs were incubated with phi29 polymerase (New England Biolabs, Ipswich, MA, USA) and reaction buffer (0.75 U of phi29 polymerase, 0.1 mg/ml bovine serum albumin, 0.2 mM deoxynucleoside triphosphate mix, and 1× phi29 buffer) at 30°C for 12 h and then at 65°C for 20 min. Samples were loaded into slot blots and then hybridized with 32P-labeled C-probe (CCCTAA)3 under native conditions to measure the amplified C-circle level.
Phi29 polymerase
Manufactured by New England Biolabs
Sourced in United States
Phi29 polymerase is a DNA-dependent DNA polymerase enzyme isolated from the Bacillus subtilis bacteriophage Phi29. It exhibits high processivity and strand-displacement activity, enabling it to efficiently amplify long DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Phi29 reaction buffer, by New England Biolabs (7 mentions)
Bovine serum albumin (bsa), by New England Biolabs (7 mentions)
Dntps, by New England Biolabs (6 mentions)
T4 dna ligase, by New England Biolabs (5 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (3 mentions)
Lambda exonuclease, by Illumina (2 mentions)
Ampligase, by Illumina (2 mentions)
Simpliamp, by Thermo Fisher Scientific (2 mentions)
Hiseq x sequencer, by Illumina (2 mentions)
Quant it picogreen assay, by Thermo Fisher Scientific (2 mentions)
Phi29 random hexamer primer, by New England Biolabs (2 mentions)
Novaseq s4, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Phi29 polymerase, by Eppendorf (2 mentions)
Mastercycler gradient, by Eppendorf (2 mentions)
Hybond n membrane, by Cytiva (2 mentions)
Random hexamer, by Thermo Fisher Scientific (2 mentions)
Mj thermal cycler, by Bio-Rad (1 mentions)
Qubit dsdna high sensitivity, by Thermo Fisher Scientific (1 mentions)
23 protocols using phi29 polymerase
1
Quantifying Telomeric C-Circle Amplification
2
Whole Genome Amplification for Sequencing
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The sWGA method was performed on genomic DNA from unfiltered samples according to published protocols [9 (link)]. The sWGA reaction was performed in 0.2 mL PCR-tubes, containing 10 ng of template genomic DNA, 1 × BSA (New England Biolabs), 1 mM dNTPs (New England Biolabs), 2.5 µM of each amplification primer (Additional file 1 : Table S2), 1 × Phi29 reaction buffer (New England Biolabs), 30 units of Phi29 polymerase (New England Biolabs), and molecular biology grade water to reach a final reaction volume of 50 µL. The reaction was carried out on a thermocycler with the following step-down program: 5 min at 35 °C, 10 min at 34 °C, 15 min at 33 °C, 20 min at 32 °C, 30 min at 31 °C, 16 h at 30 °C, then heating for 15 min at 65 °C to inactivate the Phi29 polymerase before cooling to 4 °C. Amplified products were quantified using the Qubit® dsDNA high sensitivity kit (Thermo Fisher Scientific) to determine whether there was at least 500 ng of product for sequencing. Amplified products were cleaned using Agencourt Ampure XP beads (Beckman Coulter) as follows: 1.8 volumes of beads were added to 1 volume of amplified products, briefly mixed, and then incubated for 5 min at room temperature. A magnetic rack was used to capture the DNA binding beads. The DNA binding beads were then washed twice using 200 µL of 80% ethanol and eluted with 60 µL of EB buffer.
3
C-Circle Assay for Telomere Analysis
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The C-circle assay was performed as described in Henson et al. (44 (link)). Briefly, genomic DNA was digested by RsaI and HinfI overnight at 37°C. The digested DNA was precipitated using ethanol and resuspended in H2O. Digested DNA was then amplified using Phi29 polymerase (New England Biolabs M0269S). The amplified product was then immobilized to an Amersham Hybond N+ membrane using a slot dot blot apparatus. C-circle products were then detected by hybridization with γ-32P-(CCCTAA)4 telomeric probes.
4
Telomeric Repeat Detection Protocol
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Was performed according to6 (link). Briefly, 25 ng of genomic DNA, digested with AluI and MboI, were incubated for 12 h at 30 °C with 7.5 Units of Phi29 polymerase (NEB M0269) in Phi29 NEB buffer 1X, supplemented with dNTPs 0.37 mM each, in a final volume of 20 µl. The enzyme was inactivated by heating to 65 °C for 20 minutes and the reaction was blotted onto a Hybond-X membrane. Telomeric repeats were detected using the TTAGGG repeats probe described above.
5
Selective Whole Genome Amplification Protocol
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The sWGA reaction was performed in 0.2 ml PCR-tubes or plates. The reaction (50 µl total volume) containing at least 5 ng of template DNA, 1× BSA (New England Biolabs), 1 mM dNTPs (New England Biolabs), 2.5 µM of each amplification primer, 1× Phi29 reaction buffer (New England Biolabs), and 30 units of Phi29 polymerase (New England Biolabs), was placed in a PCR machine (MJ thermal Cycler, Bio-Rad) programmed to run a “stepdown” protocol consisting of 35 °C for 5 min, 34 °C for 10 min, 33 °C for 15 min, 32 °C for 20 min, 31 °C for 30 min, 30 °C for 16 h then heating at 65 °C for 15 min to inactivate the enzymes prior to cooling to 4 °C. Once the product was amplified, it was quantified using Qubit® dsDNA high sensitivity (Thermo Fisher Scientific) to determine whether there was enough material for sequencing—minimum required is 500 ng of product. Standard whole genome amplified (WGA) products of the test samples were also sequenced as control to determine the extent of enrichment [12 (link)].
6
Padlock Probe-based microRNA Detection
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Tris buffer solution (1 mol L−1, pH 8.0), dNTP mixture (25 mmol L−1) and RNase inhibitor were purchased from Solarbio Life Sciences (China). Sodium chloride and magnesium chloride were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). The microRNA, padlock probe and three oligonucleotides (Table 1 ) were synthesized and HPLC-purified by Sanggon Biotech Co., Ltd. (Shanghai, China). Ultrapure water was purchased Dongsheng Biotech Co., Ltd. (Guangzhou, China). T4 DNA ligase, phi29 polymerase, exonuclease I (EXO I) and bovine serum albumin (BSA) were purchased from New England Biolabs (Ipswich, MA, USA).
7
DNA Fragment Circularization and Amplification
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Example 1
A protocol suitable for performing the method illustrated in
8
Plasmodium DNA Extraction, Amplification, and Sequencing
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Qiagen DNA mini kits were used to extract and purify the genomic DNA and Quant-iT™ PicoGreen® Assay (Invitrogen) was used to quantify the amount of DNA. For samples with less than 50 ng DNA obtained, whole genome amplification (WGA) was performed before NGS library preparation. WGA reactions were performed following Nair et al. (Nair et al., 2014 (link)). Each 25 μL reaction contained at least 5 ng of Plasmodium DNA, 1× BSA (New England Biolabs), 1 mM dNTPs (New England Biolabs), 3.5 μM of Phi29 Random Hexamer Primer, 1× Phi29 reaction buffer (New England Biolabs), and 15 units of Phi29 polymerase (New England Biolabs). We used a PCR machine (SimpliAmp, Applied Biosystems) programmed to run a “stepdown” protocol: 35°C for 10 min, 34°C for 10 min, 33°C for 10 min, 32°C for 10 min, 31°C for 10 min, 30°C for 6 h then heating at 65°C for 10 min to inactivate the enzymes prior to cooling to 4°C. Samples were cleaned with AMPure XP Beads (Beckman Coulter) at a 1:1 ratio.
Next generation sequencing (NGS) libraries were constructed using 50–100 ng DNA or WGA product following the KAPA HyperPlus Kit protocol with 3-cycle of PCR. All libraries were sequenced at 150 bp pair-end using Illumina Novaseq S4 or Hiseq X sequencers. All bulk were sequenced to a minimum coverage of 100×.
Next generation sequencing (NGS) libraries were constructed using 50–100 ng DNA or WGA product following the KAPA HyperPlus Kit protocol with 3-cycle of PCR. All libraries were sequenced at 150 bp pair-end using Illumina Novaseq S4 or Hiseq X sequencers. All bulk were sequenced to a minimum coverage of 100×.
9
Whole Genome Amplification and Sequencing of Plasmodium
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We used Qiagen DNA mini kit to extract and purify the genomic DNA, and Quant-iT™ PicoGreen® Assay (Invitrogen) to quantify the amount of DNA. For samples with less than 50ng DNA obtained, whole genome amplification (WGA) was performed before NGS library preparation. WGA reactions were performed following Nair et al (Nair et al., 2014). Each 25 μl reaction contained at least 5ng of Plasmodium DNA, 1× BSA (New England Biolabs), 1 mM dNTPs (New England Biolabs), 3.5 μM of Phi29 Random Hexamer Primer, 1× Phi29 reaction buffer (New England Biolabs), and 15 units of Phi29 polymerase (New England Biolabs). We used a PCR machine (SimpliAmp, Applied Biosystems) programmed to run a “stepdown” protocol: 35 °C for 10 min, 34 °C for 10 min, 33 °C for 10 min, 32 °C for 10 min, 31 °C for 10 min, 30 °C for 6 h then heating at 65 °C for 10 min to inactivate the enzymes prior to cooling to 4 °C. Samples were cleaned with AMPure XP Beads (Beckman Coulter) at a 1:1 ratio. We constructed next generation sequencing (NGS) libraries using 50–100 ng DNA or WGA product following the KAPA HyperPlus Kit protocol with 3-cycle of PCR. All libraries were sequenced at 150bp pair-end using Illumina Novaseq S4 or Hiseq X sequencers. We sequenced all bulk samples to a minimum coverage of 100×.
10
DNA Detection Using Molecular Beacons
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T4 DNA ligase and phi29 polymerase (10 units per μL) were obtained from New England Biolabs (Beijing, China). Nb.Mva1269I (10 units per μL) was bought from Fermentas. Deoxyribonucleoside triphosphates (dNTPs, 100 mM), NaCl and KCl were bought from Beijing DingGuo Biotech. Co., Ltd. All solutions were prepared using ultrapure water. The DNA sequences were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. The molecular beacon (MB) modified with 5′-FAM and 3′-dabcyl was purified through high-performance liquid chromatography (HPLC). Other DNA sequences without modification were purified through denaturing polyacrylamide gel electrophoresis (PAGE). The sequences of the oligonucleotide probes used in this work are listed in Table S1 and Fig. S1.† Five single-stranded oligonucleotides (H1, H2, H3, H4, H5) were hybridized with each other to form the track with three protruding single-stranded branches (A, B, C). The sequences of the RCA products of C1 and C2 are complementary to the underlined sequences of the MB.
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