The 10,051 bp biotin-labeled dsDNA was generated by PCR39 (link) using the TaKaRa LA Taq kit (Takara Bio, Inc). The substrate DNA was lambda phage DNA (New England Biolabs) and the primers were as follows. “Lambda10kbForward”: (biotin TEG)-5′-CTGATGAGTTCGTGTCCGTACAACTGGCGTAATC and “Lambda10kbReverse”: 5′-ATACGCTGTATTCAGCAACACCGTCAGGAACACG (IDT DNA, Inc.).
Lambda phage dna
Manufactured by New England Biolabs
Sourced in Japan
Lambda phage DNA is a linear double-stranded DNA molecule that serves as the genetic material of the lambda bacteriophage. It has a size of approximately 48.5 kilobase pairs and contains the necessary genetic information for the phage's replication and infection of host bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Takara la taq kit, by Takara Bio (1 mentions)
Typhoon 9500 scanner, by GE Healthcare (1 mentions)
P4005, by Merck Group (1 mentions)
La taq dna polymerase, by Takara Bio (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Label it cy 5 labelling kit, by Mirus Bio (1 mentions)
Suncoast yellow fluorescent polymer nanospheres, by Bangs Laboratories (1 mentions)
Ecorv, by New England Biolabs (1 mentions)
Linear pbr322 plasmid dna, by New England Biolabs (1 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (1 mentions)
8 protocols using lambda phage dna
1
Biotin-Labeled dsDNA Amplification from Lambda Phage
2
Histone Exchange Reaction Assay
3
Histone Exchange Assay for SWR Chromatin Remodeling
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The histone exchange assay was conducted using a protocol modified from (32 (link)). Each reaction is 25 μl and is composed of three parts. Part A, which constitutes 60% of the reaction volume, contains 4 nM purified SWR in 25 mM HEPES–KOH (pH 7.6), 0.5 mM EGTA, 0.1 mM EDTA, 5 mM MgCl2, 0.17 μg/μl BSA, 50 mM NaCl, 10% glycerol, 0.02% NP-40. Part B, which constitutes 20% of the reaction volume, contains 75 nM Cy3-labeled AA nucleosome and 550 nM H2A.Z–H2BFL dimers in 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl. Part C is 1 mM ATP and represents 20% of the reaction volume. Part A and Part B were mixed together before Part C was added to initiate the reaction. The reaction was left at room temperature (∼22°C) for the indicated times before they were quenched by the addition of 62.5 ng of lambda phage DNA (New England Biolabs). Five microliter of Nap1 at 3.5 μM in buffer S [70% (w/v) sucrose, 10 mM Tris–HCl (pH 7.8), 1 mM EDTA] was added to the reaction immediately before resolving the nucleosomes on 6% polyacrylamide/0.5× TBE gels. In-gel Cy3 fluorescence was detected by a Typhoon 9500 scanner (GE Healthcare) and densitometry was performed using the ImageQuant software. For the Swc5 rescue experiments, Swc5 and/or H2A–H2B dimer were added to the Part A and B mixture at the indicated final concentrations before adding in Part C.
4
DNA-Protamine Binding Characterization
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We purchased salmon protamine (P4005, 1.1 kDa, Sigma Aldrich) which contained 32 residues and had a +21 charge (21 arginine residues). We dissolved the protamine and stored aliquots at -20°C.
DNA of varying lengths [L = 25 nm (75 bp), 50 nm (150 bp), 105 nm (309 bp) and 217 nm (639 bp)] was made via Polymerase Chain Reaction (PCR) using standard protocols (29 (link),32–34 (link)), lambda phage DNA (New England Biolabs, N3011L) as a template, and LA Taq DNA polymerase (TaKaRa Bio, RR002). To biochemically tether the DNA to the surface of the cover slip and the streptavidin-coated polystyrene bead, one primer was labelled with a digoxigenin molecule, while the other was labelled with a biotin molecule (Integrated DNA Technologies). To purify the DNA, we performed gel electrophoresis using orange loading dye (B7022S, New England Biolabs) and standard protocols for short DNA molecules (35 ), then purified the DNA via gel extraction (QIAquick Gel Extraction Kit, Qiagen). We quantified the final concentration using a spectrophotometer (Nanodrop One, ThermoFisher Scientific).
DNA of varying lengths [L = 25 nm (75 bp), 50 nm (150 bp), 105 nm (309 bp) and 217 nm (639 bp)] was made via Polymerase Chain Reaction (PCR) using standard protocols (29 (link),32–34 (link)), lambda phage DNA (New England Biolabs, N3011L) as a template, and LA Taq DNA polymerase (TaKaRa Bio, RR002). To biochemically tether the DNA to the surface of the cover slip and the streptavidin-coated polystyrene bead, one primer was labelled with a digoxigenin molecule, while the other was labelled with a biotin molecule (Integrated DNA Technologies). To purify the DNA, we performed gel electrophoresis using orange loading dye (B7022S, New England Biolabs) and standard protocols for short DNA molecules (35 ), then purified the DNA via gel extraction (QIAquick Gel Extraction Kit, Qiagen). We quantified the final concentration using a spectrophotometer (Nanodrop One, ThermoFisher Scientific).
5
DNA Labeling and Nanosphere Characterization
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Supercoiled ColE1 (6.6 kbp) DNA was obtained from Nippon Gene (Toyama, Japan) whereas the lambda phage DNA was obtained from New England Biolabs (Hitchin, UK). The restriction enzyme SmaI and the digestion buffer were obtained from New England Biolabs and were used to prepare the linear form of the ColE1 DNA. The DNA molecules were covalently labelled with Cy5 using a Label IT Cy 5 labelling kit obtained from Mirus Bio (Madison, WI, USA).
Suncoast yellow fluorescent polymer nanospheres (excitation/emission maxima 540/600 nm) of nominal size (0.19 μm; 2.653 × 1012 nanospheres ml−1) were purchased from Bangs Laboratories, Inc. (Fishers, IN, USA). The nanospheres were diluted with 70% glycerol in 10 mM TRIS buffer (pH 8) to yield a concentration of 1.5 × 106 nanospheres ml−1.
Suncoast yellow fluorescent polymer nanospheres (excitation/emission maxima 540/600 nm) of nominal size (0.19 μm; 2.653 × 1012 nanospheres ml−1) were purchased from Bangs Laboratories, Inc. (Fishers, IN, USA). The nanospheres were diluted with 70% glycerol in 10 mM TRIS buffer (pH 8) to yield a concentration of 1.5 × 106 nanospheres ml−1.
6
Biotinylated DNA Synthesis via PCR
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To produce
∼5 kb DNA
with biotin attached at one end, we performed PCR according to the
manufacturer’s recommendations (PCR Extender System, QuantaBio,
Beverly, Massachusetts) using Lambda phage DNA as a template (New
England Biolabs Inc., Ipswich, Massachusetts), a biotinylated primer
and an unmodified primer (forward 5′-biotinTEG-CTGATGAGTTCGTGTCCGTACAACTGGCGTAATC-3′,
reverse 5′-GTTTGTACTCCAGCGTCTCATCTTTATGCGCC-3′,
Integrated DNA Technologies). This produced a single band in a conventional
agarose gel electrophoresis experiment at the expected 5031 bp. The
cleaned PCR product was combined with biotin-plugged divalent streptavidin,
where the oligonucleotides of the two plugging molecules had been
cleaved off with 5 min UV irradiation, and incubated overnight in
100 mM sodium phosphate buffer pH 6.5. Products of the incubation
were visualized on a 0.5% agarose gel using staining with ethidium
bromide.
∼5 kb DNA
with biotin attached at one end, we performed PCR according to the
manufacturer’s recommendations (PCR Extender System, QuantaBio,
Beverly, Massachusetts) using Lambda phage DNA as a template (New
England Biolabs Inc., Ipswich, Massachusetts), a biotinylated primer
and an unmodified primer (forward 5′-biotinTEG-CTGATGAGTTCGTGTCCGTACAACTGGCGTAATC-3′,
reverse 5′-GTTTGTACTCCAGCGTCTCATCTTTATGCGCC-3′,
Integrated DNA Technologies). This produced a single band in a conventional
agarose gel electrophoresis experiment at the expected 5031 bp. The
cleaned PCR product was combined with biotin-plugged divalent streptavidin,
where the oligonucleotides of the two plugging molecules had been
cleaved off with 5 min UV irradiation, and incubated overnight in
100 mM sodium phosphate buffer pH 6.5. Products of the incubation
were visualized on a 0.5% agarose gel using staining with ethidium
bromide.
7
Characterization of SYBR Gold Dye
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For MT measurements, we use a 7.9-kbp DNA construct, prepared as described previously (24) . The construct is generated by ligating handles (~600 bp) with either multiple biotin or multiple digoxigenin moieties fragments to an unmodified central DNA segment 7.9 kbp in length. Fluorescence intensity measurements of SYBR Gold are recorded in the presence of linear pBR322 plasmid DNA (NEB), which is produced by restriction of supercoiled circular pBR322 using restriction enzyme EcoRV (NEB) according to the protocol provided by the manufacturer. Completion of the linearization reaction was validated by agarose gel electrophoresis. Absorption, excitation and emission spectra of SYBR Gold are recorded in the presence of lambda phage DNA (NEB), which was dialyzed against phosphate buffered saline (PBS) prior to use.
8
Fabrication of Semiflexible Magnetic Filaments
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The semiflexible filaments are fabricated using superparamagnetic colloidal particles linked together with double stranded DNA (dsDNA). The particles are streptavidin functionalized polystyrene spheres (Dynabeads® MyOne™ Streptavidin C1, Life Technologies Corp.). The mean diameter of the particles is 2a = 1.05±0.1 μm, density is 1.8 g/cm 3 , and effective volumetric magnetic susceptibility is χ eff = 1.38, as provided by the manufacturer [26] . DNA fragments of 1250, 2000 and 4000 base pairs (bp) are biotinylated on the 5' ends. They are formed by lysing lambda-phage DNA (New England Biolabs, Ipswich, MA) using standard polymerase chain reaction (PCR) procedures [27] . The superparamagnetic filaments are prepared inside glass chambers filled with aqueous solution (10 mM phosphate buffer solution) using methods previously described [27] . The colloidal particles are denser than the aqueous media and rapidly sediment to the bottom of the sample chamber; therefore, the filaments exist in a quasitwo-dimensional environment. Filament flexibility is able to be tuned by altering the length of DNA linkers (changing the length of springs) or adjusting the field strength for linking (changing interparticle distance).
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