Trace nucleic acid sequencing pretreatment method based on magnetic bead coating and kit
A nucleic acid sequencing and magnetic bead technology, applied in the field of molecular biology, can solve the problems of scarcity of DNA, difficulty in reaching the total amount of DNA, inability to directly read DNA sequences, etc., to reduce DNA damage, high success rate and low cost Effect
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Embodiment 1
[0097] 1. Reagent preparation:
[0098] Ampure XP Beads (Beckman), absolute ethanol (Sinopharm), polyethylene glycol 8000 (Sigma), sodium chloride (Sinopharm), Tris (Sinopharm), HCl (Sinopharm), Qubit dsDNA HS Assay (Life Technologies), 10X T4 DNA ligation buffer (NEB), bovine serum albumin (1mg / mL, NEB), ATP (10mM, NEB), dNTPs (10mM, NEB), T4 phosphorylated kinase (NEB), T4 DNA polymerase (NEB), Klenow Large Fragment Polymerase (NEB), 10×Klenow Polymerization Buffer (NEB), Adenine (1mM, NEB), Klenow Polymerase (exo-, NEB), 2×DNA Ligation Buffer (NEB), DNA Ligase (NEB), Illumina Sequencing Adapter (Bioo Scientific), 2×KAPA HiFi Ready Mix (KAPA Biosystem), Illumina Primer Mix (IDT).
[0099] Reagent I: pH 8.0, the composition is: 5uL of 10X T4 DNA ligation buffer, 5uL of bovine serum albumin (1mg / mL), 5uL of ATP (10mM), 2uL of dNTPs (10mM), 5uL of T4 phosphorylated kinase, T4 DNA polymerization Enzyme 5uL, Klenow large fragment polymerase 1uL, ultrapure water 22uL.
[0100] ...
Embodiment 2
[0139] 1. Reagent preparation:
[0140] Ampure XP Beads (Beckman), absolute ethanol (Sinopharm), polyethylene glycol 8000 (Sigma), sodium chloride (Sinopharm), Tris (Sinopharm), HCl (Sinopharm), Qubit dsDNA HS Assay (Life Technologies), 10X T4 DNA ligation buffer (NEB), bovine serum albumin (1mg / mL, NEB), ATP (10mM, NEB), dNTPs (10mM, NEB), T4 phosphorylated kinase (NEB), T4 DNA polymerase (NEB), Klenow Large fragment polymerase (NEB), 10×Klenow polymerization buffer (NEB), adenine (1mM, NEB), Klenow polymerase (exo-, NEB), 2×DNA ligation buffer (NEB), DNA ligase ( NEB), Illumina sequencing adapter (Bioo Scientific), 2×KAPA HiFi Ready Mix (KAPA Biosystem), Illumina Primer Mix (IDT).
[0141] Reagent I: pH 8.0, the composition is: 5uL of 10X T4 DNA ligation buffer, 5uL of bovine serum albumin (1mg / mL), 5uL of ATP (10mM), 2uL of dNTPs (10mM), 5uL of T4 phosphorylated kinase, T4 DNA polymerization Enzyme 5uL, Klenow large fragment polymerase 1uL, ultrapure water 22uL.
[0142]...
Embodiment 3
[0181] 1. Reagent preparation:
[0182] Ampure XP Beads (Beckman), absolute ethanol (Sinopharm), polyethylene glycol 8000 (Sigma), sodium chloride (Sinopharm), Tris (Sinopharm), HCl (Sinopharm), Qubit dsDNA HS Assay (Life Technologies), 10X T4 DNA ligation buffer (NEB), bovine serum albumin (1mg / mL, NEB), ATP (10mM, NEB), dNTPs (10mM, NEB), T4 phosphorylated kinase (NEB), T4 DNA polymerase (NEB), Klenow Large fragment polymerase (NEB), 10×Klenow polymerization buffer (NEB), adenine (1mM, NEB), Klenow polymerase (exo-, NEB), 2×DNA ligation buffer (NEB), DNA ligase ( NEB), Illumina sequencing adapter (Bioo Scientific), 2×KAPA HiFi Ready Mix (KAPA Biosystem), Illumina Primer Mix (IDT).
[0183] Reagent I: pH 8.0, the composition is: 10X T4 DNA ligation buffer 5uL, bovine serum albumin (1mg / mL) 5uL, ATP (10mM) 5uL, dNTPs (10mM) 2uL, T4 phosphorylated kinase 5uL, T4 DNA polymerization Enzyme 5uL, Klenow large fragment polymerase 1uL, ultrapure water 22uL.
[0184] Reagent II: th...
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