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λ dna hindiii

Manufactured by New England Biolabs

λ DNA/HindIII is a DNA ladder product offered by New England Biolabs. It consists of fragments of λ DNA digested with the HindIII restriction enzyme, providing a set of DNA fragments of known sizes for use as molecular weight markers in gel electrophoresis.

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2 protocols using λ dna hindiii

1

DNA Samples for Biopolymer Translocation

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For the translocated biopolymer, several DNAs have been used: λ DNA (48.5 kbp, ThermoFisher Scientific), λ DNA/HindIII (125 bp to 23,130 bp, average 14.3 kbp, New England Biolabs), T4 GT7 DNA (166 kbp, NIPPON GENE), Φ X174 RF II DNA (5.4 kbp, New England Biolabs), pNEB206A linearized (2.7 kbp, New England Biolabs), pCLIPf-H2B (6.2 kbp, circular DNA, New England Biolabs), pKLAC2 (9.1 kbp, circular DNA, New England Biolabs), and DNA ladder (100 bp to 1,517 bp, average 710 bp, New England Biolabs). The concentration of DNA was kept constant at 4.9 nM (in phosphate groups equivalent) in buffer Tris-KCl (10 mM) and (ethylenedinitrilo)tetraacetic acid (1 mM) at pH 7.6 (25 C). The DNAs have been fluorescently labeled with YOYO-1 (ThermoFisher Scientific) at 3.0 nM (about one YOYO-1 molecule available per DNA base).
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2

Plasmid DNA Isolation and Cloning

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Plasmid DNA was isolated by alkaline lysis (11 (link)). DNA was digested with restriction enzymes according to the manufacturer’s instructions, and fragments were separated through 0.7% agarose (Bioline) in 1 × Tris-acetate-EDTA (TAE) buffer. The size standards were a 1-kb ladder and λ DNA-HindIII (New England Biolabs).
PCRs were performed as previously described (11 (link)). The sequences for all primers used in this study are listed in Table 3. PCR products were purified for sequencing via gel extraction using an EconoSpin DNA column (Epoch Life Sciences). Routine sequencing was performed at the Australian Genome Research Facility (Sydney).
Cloning was performed as previously described (11 (link)), with minor modifications. All ligations were performed at a 3:1 M ratio of insert to vector with 40 U of T4 DNA ligase (New England Biolabs) in a final concentration of 1× ligase buffer. Chemically competent E. coli DH5α cells were prepared using a CaCl2 method, and transformation was performed via heat shock.
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