Longamp polymerase

Manufactured by New England Biolabs

Sourced in United States

LongAmp polymerase is a thermostable DNA polymerase designed for long-range PCR amplification. It is capable of generating high-fidelity DNA fragments up to 30 kb in length.

Automatically generated - may contain errors

Lab products found in correlation

Topo vector, by Thermo Fisher Scientific (2 mentions) Primerstar gxl, by Takara Bio (2 mentions) Protoscript 2, by New England Biolabs (1 mentions) Viral rna isolation kit, by Qiagen (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Hifi assembly master mix, by New England Biolabs (1 mentions) Pgem t easy, by New England Biolabs (1 mentions) Monarch gel extraction kit, by New England Biolabs (1 mentions) Protoscript 2 reverse transcriptase, by New England Biolabs (1 mentions)

Close spelling variants of this product

LongAmp Taq DNA polymerase kit LongAmp high-fidelity Taq-DNA polymerase LongAmp Taq polymerase LongAmp® Hot Start Taq DNA polymerase LongAmp DNA polymerase LongAmp Taq DNA Polymerase

7 protocols using longamp polymerase

Genomic DNA Extraction and Amplification for Single-Cell and Bulk Sequencing

Cited 1 time

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

For single-cell clones or bulk sequencing, genomic DNA (gDNA) was extracted by quick extract (Lucigen). PCR amplification was performed with LongAmp Polymerase (NEB) or PrimerStar GXL (Takara). Primer pairs flanking the upstream cut site or downstream cut site were used. Amplicons were verified by gel extraction and Sanger sequencing. Amplicons from bulk sequencing were cloned into the TOPO vector (Invitrogen) before Sanger sequencing.
The frequency of homozygous and heterozygous integration in HeLa cells was determined by knocking-in mCherry and miRFP670 simultaneously. By measuring mCherry⁺, miRFP670⁺ and double positive cells, the homozygous knock-in could be calculated [7 (link)].
We used modified ICE analysis for deconvolution of amplicon Sanger trace data derived from unsorted Replace targeted cells [45 (link)]. The amplicons were made using a primer on the donor sequence and a primer on the genomic sequence flanking the ligated site. The amplicon was cloned into the TOPO vector and individual cloned alleles were Sanger sequenced along with the mixed PCR product. A cloned colony with Replace inserts without any InDels was identified, and these Sanger trace data were used as the ‘wild-type’ reference in ICE analysis.

Danner E., Lebedin M., de la Rosa K, & Kühn R. (2021). A homology independent sequence replacement strategy in human cells using a CRISPR nuclease. Open Biology, 11(1), 200283.

+ Open protocol

+ Expand

Biallelic BREX Locus Verification

Cited 2 times

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

The BREX engineered locus sequence was verified in two ways. First, PCR reactions (primers oJZ_241/242, S2 File) with LongAmp polymerase (New England Biolabs; Ipswich, MA, USA), purified and sequenced (Sanger). Second, PacBio sequencing reads from the methylation analysis was aligned with the in silico design of the engineered locus.
DNA motifs and degree of modification were generated using InterPulse Duration (IPD) Ratios analyzed with RS_Modification_and_Motif_Analysis from Pacific Biosciences as in [93 (link),94 (link)]. PBMotStat, a component of REBASE TOOLS [95 (link)] was used to calculate the % of methylated sites with IPD > 2 for specific sites.
For the partially methylated strains, a.gff file of the methylation level of each base of the genome was downloaded from SMRT Link, filtered to keep only significantly methylated sites, and uploaded in Geneious Prime on the reference genome (S5 File).

Zaworski J., Dagva O., Brandt J., Baum C., Ettwiller L., Fomenkov A, & Raleigh E.A. (2022). Reassembling a cannon in the DNA defense arsenal: Genetics of StySA, a BREX phage exclusion system in Salmonella lab strains. PLoS Genetics, 18(4), e1009943.

+ Open protocol

+ Expand

Amplifying SAV and ISAV Genomes

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

Total RNA was extracted from SAV and ISAV samples (Table 2) using a phenol-chloroform extraction method, except for the SAV6 sample, which was extracted using a Viral RNA Isolation kit (Qiagen). cDNA was synthesised using Protoscript II (New England Biolabs) reverse transcriptase and a mix of random hexamer and oligo dT (dT₂₃VN) primers (New England Biolabs) as per the manufacturers’ instructions. First-strand cDNA was used as template for long-range PCR reactions.
To amplify the SAV1/6 genomes, degenerate PCR primers targeting three ~4 kb overlapping amplicons were designed in regions of the genome conserved in the five subtypes where sequence data is available (Table 1). PCR was conducted using LongAmp polymerase (New England Biolabs) with cycling conditions as follows: 30 s at 94 °C, followed by 35 cycles of 15 s at 94 °C, 1 min at 56 °C and 3 min 50 s at 65 °C, with a final extension for 10 min at 65 °C. ISAV segments 5 and 6 were amplified using the same approach and primers designed to conserved 5′ and 3′ regions of segment 5/6 (Table 2) under the same conditions, except that the PCR extension time was 2 min 30 s. PCR products were visualised on a 1% agarose gel, purified using QIAquick Gel Extraction Kit (Qiagen) and stored at −80 °C until sequencing.

Gallagher M.D., Matejusova I., Nguyen L., Ruane N.M., Falk K, & Macqueen D.J. (2018). Nanopore sequencing for rapid diagnostics of salmonid RNA viruses. Scientific Reports, 8, 16307.

+ Open protocol

+ Expand

Transposable Element Insertion Sequencing

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

The transposable element (TE) insertion was amplified from s¹ genomic DNA using LongAmp polymerase (New England Biolabs) with tailed primers gibpg7-Yippee-5′-Region-F1 gcggccgcgggaattcgattCCGGGCAGCCACGCAAGGATTGCAT and gibpg6-Yippee-5′region-R2 ccgcgaattcactagtgattGGTCAGGTGTCCGGTGTCAGGG. The ∼8 kbp PCR band was gel purified and assembled into pGem-T-Easy using HiFi Assembly Master Mix (New England Biolabs). Three clones were fully sequenced using Oxford Nanopore technology by Plasmidsaurus (Eugene, OR), then aligned to generate a consensus sequence.

Dean D.M., Deitcher D.L., Paster C.O., Xu M, & Loehlin D.W. (2022). “A fly appeared”: sable, a classic Drosophila mutation, maps to Yippee, a gene affecting body color, wings, and bristles. G3: Genes|Genomes|Genetics, 12(5), jkac058.

+ Open protocol

+ Expand

Efficient Sequencing of DRB Alleles

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

DRB alleles were amplified by long-range PCR (Long AMP polymerase, New England Biolabs) of genomic DNA. In order to
determine the nucleotide sequences, two fragments overlapping in the highly polymorphic exon 2 region were amplified (Fig. 1). The primers were designed on the basis of examination of DRB sequences available
in the IMGT database [6 ] and Ensembl [20 (link)] with
the aid of Integrated DNA Technologies OligoAnalyzer v3.1 software. 0.2 M Trehalose was introduced into PCR reaction to obtain
reliable and efficient amplification [21 (link)].

Barsakis K., Babrzadeh F., Chi A., Mallempati K., Pickle W., Mindrinos M, & Fernández-Viña M.A. (2019). Complete nucleotide sequence characterization of DRB5 alleles reveals a homogeneous allele group that is distinct from other DRB genes. Human immunology, 80(7), 437-448.

+ Open protocol

+ Expand

Amplification and Sequencing of Salmonid Alphaviruses

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

Twenty-three naturally SAV-infected heart tissues of Atlantic salmon and one heart tissue from rainbow trout stored in RNAlater were obtained from PHARMAQ Analytiq (Zoetis) (Table 1). Samples were selected from across as wide a geographic range as possible and were sampled between 2016 and 2019. Total RNA was extracted from each sample using a phenol-chloroform approach, and integrity assessed using gel electrophoresis. cDNA was synthesized using Protoscript II Reverse Transcriptase (New England Biolabs) and a mix of random hexamer and anchored-dT (dT₂₃VN) primers. First strand cDNA was used as template for PCR reactions.
Viral genomes were amplified in six overlapping PCR amplicons of roughly 2 kb in length (primer sequences available in Table 2) using LongAmp polymerase (New England Biolabs) with the following PCR cycling conditions: 30 s at 94°C, followed by 35 cycles of 15 s at 94°C, 1 min at 56°C and 2 min 15 s at 65°C, with a final extension for 10 min at 65°C. Primers were designed in regions of the SAV genome conserved between SAV2 and SAV3 (Table 2). All PCR products were visualized on a 1% agarose gel before being excised and purified using Monarch Gel Extraction Kit (New England Biolabs), eluted in 10 μl of elution buffer and stored at −80°C until sequencing.

Gallagher M.D., Karlsen M., Petterson E., Haugland Ø., Matejusova I, & Macqueen D.J. (2020). Genome Sequencing of SAV3 Reveals Repeated Seeding Events of Viral Strains in Norwegian Aquaculture. Frontiers in Microbiology, 11, 740.

+ Open protocol

+ Expand

CRISPR-Mediated Gene Editing Validation

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

For single cell clones or bulk sequencing genomic DNA (gDNA) was extracted by quick extract (Lucigen). PCR amplification was performed with LongAmp Polymerase (NEB) or PrimerStar GXL (Takara). Primer pairs flanking the upstream cut site or downstream cut-site were used. Amplicons were verified by gel extraction and Sanger sequencing. Amplicons from bulk sequencing were cloned into the TOPO vector (Invitrogen) before Sanger sequencing.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Frequency of homozygous and heterozygous integration in HeLa cells was determined by knocking-in mCherry and miRFP670 simultaneously. By measuring mCherry + , miRFP670 + , and double positive cells the homozygous knock-in could be calculated (7) .
We used modified ICE analysis for deconvolution of amplicon Sanger trace data derived from unsorted Replace targeted cells (37) . The amplicons were made using a primer on the donor sequence and a primer on the genomic sequence flanking the ligated site. The amplicon was cloned into the TOPO vector and individual cloned alleles were Sanger sequenced along with the mixed PCR product. A cloned colony with Replace inserts without any InDels was identified, and this Sanger trace data was used as the 'wild-type' reference in ICE analysis.

Danner E., Lebedin M., Rosa K.d.l., & Kühn R. (2020). A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease.

+ 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!