Log In Sign up
The largest database of trusted experimental protocols

Ampligase

Manufactured by Illumina
Visit Supplier
Sourced in United States

Ampligase is a thermostable DNA ligase enzyme used in various molecular biology applications. Its core function is to catalyze the formation of phosphodiester bonds between adjacent DNA fragments, enabling DNA ligation processes.

Automatically generated - may contain errors

Lab products found in correlation

Hiseq 2000, by Illumina (3 mentions) Exonuclease 3, by Illumina (2 mentions) Exonuclease 1, by Illumina (2 mentions) Phi29 polymerase, by New England Biolabs (2 mentions) Lambda exonuclease, by Illumina (2 mentions) M 280 streptavidin coated magnetic beads, by Thermo Fisher Scientific (2 mentions) Qubit dsdna hs assay, by Thermo Fisher Scientific (2 mentions) Ampligase buffer, by Illumina (2 mentions) Hemo klentaq 0.32μl, by New England Biolabs (2 mentions) Ampure xp bead, by Beckman Coulter (2 mentions) Superfrost plus slide, by Thermo Fisher Scientific (2 mentions) Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions) Templiphi, by GE Healthcare (1 mentions) Thermocycler, by Thermo Fisher Scientific (1 mentions) Pcr purification kit, by Qiagen (1 mentions) T4 pnk, by New England Biolabs (1 mentions) Dntps, by Thermo Fisher Scientific (1 mentions) Phusion hf, by New England Biolabs (1 mentions) Dpni, by New England Biolabs (1 mentions) Amplitaq dna polymerase, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Ampligase DNA ligase (4 citations) Ampligase buffer (3 citations) Thermostable Ampligase (2 citations)

17 protocols using ampligase

1

Circularization and Amplification of Genomic Fragments

Check if the same lab product or an alternative is used in the 5 most similar protocols
Genomic fragments were circularized by incubating the beads with 1× Ampligase reaction buffer, 0.25 U/µL Ampligase (Epicentre, Madison, WI, USA), and 0.1 µg/µL BSA in a total volume of 50 µL at 55℃ for 10 min. The circularized molecules were separated from the beads by incubation with 5 µL sample buffer at 95℃ for 10 min and collected with a ring magnet rack. The supernatant was incubated with 5 µL reaction buffer and 0.2 µL enzyme mix (Templiphi; GE Life Sciences, Piscataway, NJ, USA) at 30℃ for 4 h, followed by inactivation at 65℃ for 10 min.
Kim E.H., Lee S., Park J., Lee K., Bhak J, & Kim B.C. (2014). New Lung Cancer Panel for High-Throughput Targeted Resequencing. Genomics & Informatics, 12(2), 50-57.
+ Open protocol
+ Expand
2

Ligation-based DNA Detection Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
The hybridization and ligation reactions were conducted in 500 μL PCR reaction vessels using a thermocycler (Applied Biosystems). The 10× Ampligase reaction buffer, 2 U Ampligase (Epicentre, Madison, WI, USA), 1 μL of one of the probes (1 μM), and a 1.5 μL template were used as reaction mixes (5 μL total volume).
The conditions were as follows: the DNA was initially denatured at 95 °C for 3 min, followed by 20 denaturation and ligation cycles at 95 °C for 15 s and 65 °C for 5 min. After ligation, the reaction mixes were heated for 5 min at 98 °C to inactivate the enzyme and then stored in a refrigerator at 4 °C.
Zhang H., Dong K., Xiang S., Lin Y., Cha X., Shang Y, & Xu W. (2023). A Novel Cu2+ Quantitative Detection Nucleic Acid Biosensors Based on DNAzyme and “Blocker” Beacon. Foods, 12(7), 1504.
+ Open protocol
+ Expand
3

Oligonucleotide Phosphorylation and Ligation for Library Preparation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Oligonucleotides were diluted to a final concentration of 10 μM. A 3 μl aliquot of each sense oligonucleotide was phosphorylated with 6 μl 10 × PNK buffer and 3 μl T4 PNK (NEB, USA). The final volume was adjusted to 60 μl by adding distilled water (dH2O) and incubated at 37 °C overnight. Nonsense oligonucleotide pools were phosphorylated by the same protocol. After 5′ phosphorylation, 20 μl sense oligonucleotide and 20 μl nonsense oligonucleotide solutions were mixed with 5 μl 10 × Ampligase buffer, 2.5 μl Ampligase (100 U per μl, Epicentre, USA), and 2.5 μl dH2O. The reaction was performed under the following conditions: (1) initial denaturation at 95 °C for 3 min; (2) annealing at 95 °C with ramping at 0.1 °C s−1 until reaching 60 °C; (3) ligation at 60 °C for 2 h; and (4) storage at 4 °C. The reactions were then purified with a Qiagen PCR purification kit according to the manufacturer's instructions (Qiagen, USA).
For the amplification, 1 μl assembled product from the first reaction was mixed with 7 μl dH2O, 10 μl KAPA HiFi 2 × polymerase (Kapa BioSystems, USA), and 1 μl each forward and reverse primers (10 μM). The mixture was subjected to PCR under the following conditions: (1) 95 °C for 3 min; (2) 95 °C for 30 s; (3) 60 °C for 30 s; (4) 72 °C for 1 min with repetition of steps (2) through (4) for 20 cycles; (5) 72 °C for 10 min; and (6) 4 °C storage.
Cho N., Hwang B., Yoon J.K., Park S., Lee J., Seo H.N., Lee J., Huh S., Chung J, & Bang D. (2015). De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries. Nature Communications, 6, 8351.
+ Open protocol
+ Expand
4

DNA Digestion and Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols
800–1000 ng DNA, which is approximately half the amount that was recovered from the previous reaction, were digested with 2 μl Surveyor endonuclease, in the presence of 2 μl Enhancer (Mutation Detection Kit, IDT, Redwood City, CA, USA), and 20 units Ampligase (Epicentre, Madison, WI, USA) in 1X Ampligase buffer and DNA concentration of 20–25 ng/μl for 50 min at 42°C, and the products were purified.
Aggeli D., Karas V.O., Sinnott-Armstrong N.A., Varghese V., Shafer R.W., Greenleaf W.J, & Sherlock G. (2018). Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery. Nucleic Acids Research, 46(7), e42.
+ Open protocol
+ Expand
5

Targeted Genome Sequencing via MIPs

Check if the same lab product or an alternative is used in the 5 most similar protocols
Individual MIPs were column synthesized (SBS Genetech, Beijing, China). MIP capture experiments were performed with some modifications as previously described [18 (link)]. In briefly, a total of 684 probes were pooled and phosphorylations were performed with ATP and Polynucleotide Kinase (NEB, Beijing, China). Pooled probes used as a master probe mix. The genomic DNA was sheared into 300-500 bp by Bandelin sonorex AK 102 sonicator (Bandelin, Berlin, Germany) and then MIPs were added to hybridization mixture (300 ng fragmented DNA, 1× Ampligase buffer). Hybridization procedure was conducted as fellows: 94 °C for 2 min, 54 °C for 36 h. And then gap filling-in reaction was conducted as fellows: 0.3 μl Phusion DNA polymerse (NEB), 0.6 μl Ampligase (Epicentre, Madison) and 0.1 μM dNTP (NEB) was added for filling-in the gap at 54 °C for 3 h. To degrade un-circularized DNA, 0.5 μl Exonuclease I (NEB) and 0.5 μl Exonuclease III (NEB) was added to reaction mixture at 37 °C for 45 min, and then exonucleases were inactivated at 94 °C for 4 min.
Liu T., Tang C, & Shi X. (2020). Analysis of variants in Chinese individuals with primary open-angle glaucoma using molecular inversion probe (MIP)-based panel sequencing. Molecular Vision, 26, 378-391.
+ Open protocol
+ Expand
6

DNA Fragment Circularization and Amplification

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

Example 1

A protocol suitable for performing the method illustrated in FIG. 1 is as follows: 1) 10 ng of DNA is digested with 1 unit of restriction enzyme in corresponding compatible restriction enzyme buffer. The reaction is incubated in 37 C for 1 h, followed by enzymatic deactivation at 80 C for 20 min. 2) The DNA fragments are denatured to single stranded fragments at 95 C for 10 min and mixed with probes and ligase to form circles. The probe pool are added in 10 pM individual concentration along with 1 U of Ampligase (Epicentre) and incubated at 55 C for 1 h in ligase buffer. 3) 1 U Exonuclease is added to remove non-reacted probes and fragments. I U of Lambda exonuclease (Epicentre) is added at 37 C for 1 h in corresponding exonuclease buffer followed by enzyme inactivation at 80 C for 20 min. 4) The remaining circles are amplified by RCA. 1 U of phi29 polymerase (New England Biolabs) is added in corresponding phi29 buffer and nucleotides (dNTPs) at 37 C for 1 h.

US10731214B2. Nucleic acid probe and method of detecting genomic fragments (2020-08-04). Vanadis Diagnostics [SE]. Inventors: Carl Oscar Fredrik Dahl [SE], Olof John Ericsson [SE].
+ Open protocol
+ Expand
7

DNA Ligation and Capture Protocol

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

Example 2

A protocol suitable for performing the method illustrated in FIG. 2 is as follows: 1) 10 ng of DNA is digested with 1 unit of restriction enzyme in corresponding compatible restriction enzyme buffer. The reaction is incubated in 37 C for 1 h, followed by enzymatic deactivation at 80 C for 20 min. 2) The DNA fragments are denatured to single stranded fragments at 95 C for 10 min and mixed with probes and ligase to form linear ligation products. The probe pool are added in 10 pM individual concentration along with 1 U of Ampligase (Epicentre) and incubated at 55 C for 1 h in ligase buffer. 3) The ligation product is captured on magnetic streptavidin beads. To remove non-reacted probes and fragments, the solution is mixed with 10 ml M-280 streptavidin coated magnetic beads (Invitrogen) in Tris-HCl (pH 7.5), 3.5 mM EDTA and 0.07% Tween-20 in a final volume of 200 ml, and incubated at room temperature for 15 min. After incubation, the beads are collected using a ring magnet and supernatant removed.

US10731214B2. Nucleic acid probe and method of detecting genomic fragments (2020-08-04). Vanadis Diagnostics [SE]. Inventors: Carl Oscar Fredrik Dahl [SE], Olof John Ericsson [SE].
+ Open protocol
+ Expand
8

DNA Ligation Protocol for Targeted Sequencing

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

Example 2

A protocol suitable for performing the method illustrated in FIG. 2 is as follows: 1) 10 ng of DNA is digested with 1 unit of restriction enzyme in corresponding compatible restriction enzyme buffer. The reaction is incubated in 37 C for 1 h, followed by enzymatic deactivation at 80 C for 20 min. 2) The DNA fragments are denatured to single stranded fragments at 95 C for 10 min and mixed with probes and ligase to form linear ligation products. The probe pool are added in 10 pM individual concentration along with 1 U of Ampligase (Epicentre) and incubated at 55 C for 1 h in ligase buffer. 3) The ligation product is captured on magnetic streptavidin beads. To remove non-reacted probes and fragments, the solution is mixed with 10 ml M-280 streptavidin coated magnetic beads (Invitrogen) in Tris-HCl (pH 7.5), 3.5 mM EDTA and 0.07% Tween-20 in a final volume of 200 ml, and incubated at room temperature for 15 min. After incubation, the beads are collected using a ring magnet and supenatant removed.

US10240198B2. Nucleic acid probe and method of detecting genomic fragments (2019-03-26). Vanadis Diagnostics [SE]. Inventors: Carl Oscar Fredrik Dahl [SE], Olof John Ericsson [SE].
+ Open protocol
+ Expand
9

Padlock Capture for DNA Analysis

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
Padlock capture was performed as previously described1 (link),3 (link),15 (link). Briefly, each reaction was performed in 20 µl volume containing 1 unit Ampligase (A3210K, Epicentre), 1 unit Phusion High-Fidelity DNA Polymerase (M0530, New England BioLabs), 1 x Phusion High-Fidelity DNA Polymerase buffer, 10 nM dNTP and 1 ng padlock probe. Two microliters of the purified pre-PCR product and 800 ng genomic DNA were used in each reaction. Nicotinamide adenine dinucleotide (NAD+) was provided in each reaction at a final concentration of 0.5 mM.
Hong R., Chandola U, & Zhang L.F. (2017). Cat-D: a targeted sequencing method for the simultaneous detection of small DNA mutations and large DNA deletions with flexible boundaries. Scientific Reports, 7, 15701.
+ Open protocol
+ Expand
10

Targeted Genomic Sequencing via Molecular Inversion Probes

Check if the same lab product or an alternative is used in the 5 most similar protocols
Hybridization of smMIPs to genomic DNA, gap filling and ligation were performed in one 25 μl reaction of 1X Ampligase buffer (Epicentre, Madison, WI) with: 120 ng of genomic DNA, 0.32 μM dNTPs, 0.5X of Hemo KlenTaq (0.32μl) (New England Biolabs, Inc., Ipswich, MA), one unit of Ampligase (Epicentre), and MIPs. MIP concentration was based on a ratio of 667 copies of each MIP to each haploid genome copy, based on the 1X pool concentration. Reactions were incubated at 95°C for 10 min then at 60°C for 18 h. Exonuclease treatment and amplification of the captured DNA was performed as previously described6 (link). We pooled 5 μl of ~96 different barcoded libraries together and purified the pools with 0.8X AMPure XP beads (Beckman Coulter, Brea, CA) according to the manufacturer’s protocol. Libraries were resuspended in 100 μl of 1X EB (Qiagen, Valencia, CA). Pools were quantified in duplicate using the Qubit dsDNA HS Assay (Life Technologies, Grand Island, NY). Multiple libraries were combined to create the final megapools of ~192 individual capture reactions for sequencing. One lane of 101 bp paired-end reads was generated for each megapool on an Illumina HiSeq 2000 according to manufacturer’s instructions.
O'Roak B.J., Stessman H.A., Boyle E.A., Witherspoon K.T., Martin B., Lee C., Vives L., Baker C., Hiatt J.B., Nickerson D.A., Bernier R., Shendure J, & Eichler E.E. (2014). Recurrent de novo mutations implicate novel genes underlying simplex autism risk. Nature communications, 5, 5595.
+ 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?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!