High molecular weight of DNA from the L. paracasei strains was sheared in a Covaris instrument (Covaris, Woburn, MA, United States) to 10 kb fragments, and the DNA size distribution was checked on a fragment analyzer (Advanced Analytical Technologies, Ames, IA, United States). Then, 5 μg of the sheared DNA was used to prepare a SMRTbell library using the PacBio DNA Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, United States), according to the manufacturer’s recommendations. The library was sequenced using one SMRT cell v2 with C2 chemistry on a PacBio RSII system (Pacific Biosciences, Menlo Park, CA, United States) that was given a movie length of 90 min.
Pacbio dna template prep kit 2
Manufactured by Pacific Biosciences
Sourced in United States
The PacBio DNA Template Prep Kit 2.0 is a laboratory equipment designed for the preparation of DNA samples prior to sequencing on PacBio sequencing platforms. The kit provides the necessary reagents and protocols for the construction of sequencing libraries from DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Rsii sequencer, by Pacific Biosciences (2 mentions)
Pbr322, by Thermo Fisher Scientific (2 mentions)
Magbead binding kit, by Pacific Biosciences (2 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Pacbio rs 2 system, by Pacific Biosciences (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Anchored oligo dt 20 primer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using pacbio dna template prep kit 2
1
High-Molecular-Weight DNA Sequencing
2
PacBio SMRT Sequencing of cDNA Libraries
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The Poly(A+) fractions of total RNAs were quantified through use of the Qubit RNA HS Assay Kit (Life Technologies), followed by conversion to cDNAs with the SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies; the included first strand enzyme was changed to SuperScript III Reverse Transcriptase). The reverse transcription (RT) reactions were primed with Anchored Oligo(dT)20 primers (Life Technologies). The cDNAs obtained were quantified with the Qubit HS dsDNA Assay Kit (Life Technologies).
SMRTbell sequencing libraries were generated by using the PacBio DNA Template Prep Kit 2.0 and the Pacific Biosciences template preparation and sequencing protocol for Very Low (10 ng) Input 2 kb libraries with carrier DNA (pBR322, Thermo Scientific). SMRTbell templates were bound to polymerases by using the DNA polymerase binding kit XL 1.0 (part #100-150-800) and v2 primers. The polymerase-template complexes were bound to magbeads with the Pacific Biosciences MagBead Binding Kit. The SMRTBell libraries were analyzed for length and concentration through use of the Agilent 2100 Bioanalyzer. DNA sequencing was carried out with a Pacific Biosciences RS II sequencer using P5-C3 chemistry. Movie lengths were 180 min.
SMRTbell sequencing libraries were generated by using the PacBio DNA Template Prep Kit 2.0 and the Pacific Biosciences template preparation and sequencing protocol for Very Low (10 ng) Input 2 kb libraries with carrier DNA (pBR322, Thermo Scientific). SMRTbell templates were bound to polymerases by using the DNA polymerase binding kit XL 1.0 (part #100-150-800) and v2 primers. The polymerase-template complexes were bound to magbeads with the Pacific Biosciences MagBead Binding Kit. The SMRTBell libraries were analyzed for length and concentration through use of the Agilent 2100 Bioanalyzer. DNA sequencing was carried out with a Pacific Biosciences RS II sequencer using P5-C3 chemistry. Movie lengths were 180 min.
3
PacBio Sequencing of W303 Yeast Strain
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A 500 ml culture of wild-type W303 (MY7521) MATa his3 -11,15 trp1 -1, ura3 -1 (derived from strains generously provided by Rodney Rothstein, Columbia University) was grown in rich medium for ∼24 hr. Genomic DNA extraction and purification was carried out according to Burke et al. (2000) . Standard sequencing library construction was performed with Pacific Biosciences (PacBio) DNA Template Prep Kit 2.0 for one SMRT cell. The final library was sent to the University of California at Irvine for > 7 kb size selection shearing and sequencing with P6C4 chemistry.
4
PacBio Library Preparation and Sequencing
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SMRTbell libraries were generated by using the PacBio DNA Template Prep Kit 2.0 and the Pacific Biosciences template preparation and sequencing protocol for Very Low (10 ng) Input 2 kb libraries with carrier DNA (pBR322, Thermo Scientific, Waltham, MA, USA). SMRTbell templates were bound to polymerases by using the DNA polymerase binding kit XL 1.0 (part #100-150-800) and v2 primers.
Polymerase-template complexes were bound to magbeads with the Pacific Biosciences MagBead Binding Kit, and sequencing was carried out on the Pacific Biosciences RSII sequencer with C3 sequencing reagents. Movie lengths were 180 min (one movie was recorded for each SMRT Cell). Subread filtering and alignment were carried out in SMRT Pipe v2.2.0. Visualization and data analysis were performed in SMRT Analysis v2.2.0.
Polymerase-template complexes were bound to magbeads with the Pacific Biosciences MagBead Binding Kit, and sequencing was carried out on the Pacific Biosciences RSII sequencer with C3 sequencing reagents. Movie lengths were 180 min (one movie was recorded for each SMRT Cell). Subread filtering and alignment were carried out in SMRT Pipe v2.2.0. Visualization and data analysis were performed in SMRT Analysis v2.2.0.
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