T4 GT7 DNA (T4-DNA, 165.65 kbp) was purchased from Nippon Gene, Tokyo and used without further purification. The integrity of T4-DNA was verified with pulsed gel electrophoresis. No fragments of ones to tens of kbps were observed. Hfq (M = 67 kDa, hexamer) was purified from BL21(DE3)/pTE608 cells expressing untagged Hfq as previously described (50 (link)). Samples were prepared by dialyzing solutions of DNA against 10 mM Tris–HCl (T) buffer with the relevant concentration of NaCl and/or MgCl2 in micro dialyzers. For one sample series, NaCl was substituted by potassium glutamate (KGlu). Solutions of Hfq in the same buffer were also prepared. The Tris–HCl concentration is 10 mM Tris adjusted with HCl to pH 7.5 (i.e. 8.1 mM Tris–Cl and 1.9 mM Tris). The ionic strength of the buffer was calculated with the Davies equation for estimating the activity coefficients of the ions and a dissociation constant pK = 8.08 for Tris. Solutions of Hfq and DNA were subsequently mixed and incubated for 12 h at 277 K. YOYO-1 fluorescence staining dye was purchased from Invitrogen, Carlsbad, CA, USA. DNA was stained with YOYO-1 with an incubation time of 12 h and an intercalation ratio of 100 bps per dye. No anti-photo bleaching agent was used.
T4 gt7 dna
Manufactured by Nippon Gene
Sourced in Japan
The T4 GT7 DNA is a laboratory equipment designed for DNA manipulation and analysis. It is capable of performing DNA modifications such as ligation and cloning. The core function of this product is to facilitate the genetic engineering processes required for various research and development applications.
Automatically generated - may contain errors
Lab products found in correlation
Yoyo 1, by Thermo Fisher Scientific (3 mentions)
Spermine tetrahydrochloride, by Nacalai Tesque (2 mentions)
2 mercaptoethanol 2 me, by Fujifilm (1 mentions)
Tris hydrochloride acid buffer ph 7.5, by Nippon Gene (1 mentions)
λ dna, by New England Biolabs (1 mentions)
Polydimethylsiloxane, by Dow (1 mentions)
Sylgard 184, by Dow (1 mentions)
Sodium chloride (nacl), by Nacalai Tesque (1 mentions)
Milli q, by Merck Group (1 mentions)
Mgcl2, by Nacalai Tesque (1 mentions)
λ dna, by Thermo Fisher Scientific (1 mentions)
Dna ladder, by New England Biolabs (1 mentions)
Pklac2, by New England Biolabs (1 mentions)
λ dna hindiii, by New England Biolabs (1 mentions)
Carbenicillin, by Nacalai Tesque (1 mentions)
Kanamycin, by Fujifilm (1 mentions)
T7 express, by New England Biolabs (1 mentions)
Polystyrene particles, by Polysciences (1 mentions)
Te buffer, by Nippon Gene (1 mentions)
8 protocols using t4 gt7 dna
1
T4 DNA-Hfq Interaction Analysis
2
DNA Fluorescent Labeling Protocols
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Daunorubicin hydrochloride (daunomycin, DM) and the antioxidant 2-mercaptoethanol (2-ME) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Spermine tetrahydrochloride (SP) was purchased from Nacalai Tesque (Kyoto, Japan). The fluorescent cyanine dye YOYO-1 (1,10-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-[(3-methylbenzo-1,3-oxazol-2-yl) methylidene]-l,4-dihydroquinolinium] tetraiodide) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). T4 GT7 DNA (166 kbp with a contour length of 57 µm) and Tris-hydrochloride acid buffer (pH 7.5) were purchased from Nippon Gene (Tokyo, Japan). Plasmid DNA (Luciferase T7 Control DNA, 4331 bp) containing a firefly luciferase gene and a T7 RNA polymerase promoter sequence was purchased from Promega (Madison, WI, USA).
3
DNA Acquisition and PDMS Fabrication
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T4GT7 DNA was purchased from Nippon gene (Tokyo, Japan) and λ DNA was from New England Biolabs (Ipswich, MA, USA). Polydimethylsiloxane (PDMS) and its curing agent (Sylgard 184) were from Dow Corning (Midland, MI, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Linear T4GT7 DNA Characterization
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The linear T4GT7 DNA was purchased from Nippon Gene Co. Ltd. (Japan). The molar concentration of DNA shows the concentration of DNA phosphate groups (i.e. DNA monomer units, nucleotides). The fluorescent dye YOYO-1 (1,1′-(4,4,7,7-tetramethyl-4,7-diazaundeca-methylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinolinium tetraiodide) was provided by Molecular Probes (Invitrogen, Japan). NaCl, MgCl2, and spermine tetrahydrochloride were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Deionized water (Milli-Q, Millipore) was used for samples preparation. TE-KCl buffer (10 mM Tris–HCl, 1 mM EDTA, pH = 7.8) containing 10 mM of KCl was used in all experiments, except for transmission electron microscopy where Tris was replaced by HEPES.
5
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), 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).
6
Cloning and Expression of VTopoI and T4 PNK
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The gene encoding VTopoI (GenBank: L13447.1) was synthesized by Eurofins Genomics Inc (Tokyo, Japan), with codon optimization for Escherichia coli K-12, exclusion of internal BamHI and EcoRI sites, and their inclusion at the 5′- and 3′-ends of the DNA, respectively (Supplementary Sequences). The BamHI-EcoRI fragment of the gene was subcloned into the BamHI-EcoRI site of pColdTF, pColdI, and pET28a. To produce T4 polynucleotide kinase (T4 PNK), we used polymerase chain reaction (PCR) to amplify the pseT gene of T4 phage from genomic DNA (T4 GT7 DNA, Nippon Gene, Tokyo, Japan) with BamHI and EcoRI sites at the 5′ and 3′ ends, respectively (Supplementary Sequences), and cloned it into the BamHI-EcoRI site of pColdI. These constructs were introduced into T7Express (New England Biolabs, Ipswich, MA) and cultivated in Luria–Bertani broth (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) sodium chloride) with 100 µg/mL carbenicillin (for pColdTF and pColdI vector, Nacalai Tesque, Kyoto, Japan) or 50 µg/mL kanamycin (for pET28a, Fujifilm Wako Chemicals, Tokyo, Japan).
7
Polystyrene Particles for IGB Channel Characterization
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Polystyrene particles (Polyscience Inc.) were used to evaluate the IGB channel
width and to demonstrate its size tunability by changing the temperature and
dopant concentration. The d = 0.21, 0.59, 1.3,
and 3.8 μm particles were stained with Yellow Green (YG,
λex = 529 nm,
λem = 546 nm),
and the 1.3 μm particles stained with Yellow Orange (YO,
λex = 441 nm,
λem = 486 nm)
were also used. The particle diameters were evaluated by laser dynamic light
scattering using a DLS-8000 from Photal. The average diameters of the
d = 0.21, 0.59, 1.3, and
3.8 μm particles were
0.21 ± 0.04,
0.59 ± 0.11,
1.29 ± 0.26, and
3.82 ± 0.87 μm,
respectively.
The behavior of T4 GT7 DNA (166 kbp, Nippon Gene) in the IGB was also
studied. The DNA was dispersed in a TAE buffer containing 10 mM
tris(hydroxymethyl)aminomethane (tris), 0.25 mM EDTA, and 5mM acetic
acid adjusted to a pH of 2.16 or 8.41. DAPI
(4′,6-diamino-2-phenylindole) solution was then added to the DNA
solution to stain the DNA. The final concentrations of DNA and DAPI in the
sample were
4.0 × 10−13and
4.3 × 10−7 M,
respectively. Fluorescence from the stained DNA was measured with
λex = 330–385 nm
and λem ≧420 nm. All solutions
were prepared in Milli Q water. Reagents of analytical grade were used as
received.
width and to demonstrate its size tunability by changing the temperature and
dopant concentration. The d = 0.21, 0.59, 1.3,
and 3.8 μm particles were stained with Yellow Green (YG,
λex = 529 nm,
λem = 546 nm),
and the 1.3 μm particles stained with Yellow Orange (YO,
λex = 441 nm,
λem = 486 nm)
were also used. The particle diameters were evaluated by laser dynamic light
scattering using a DLS-8000 from Photal. The average diameters of the
d = 0.21, 0.59, 1.3, and
3.8 μm particles were
0.21 ± 0.04,
0.59 ± 0.11,
1.29 ± 0.26, and
3.82 ± 0.87 μm,
respectively.
The behavior of T4 GT7 DNA (166 kbp, Nippon Gene) in the IGB was also
studied. The DNA was dispersed in a TAE buffer containing 10 mM
tris(hydroxymethyl)aminomethane (tris), 0.25 mM EDTA, and 5mM acetic
acid adjusted to a pH of 2.16 or 8.41. DAPI
(4′,6-diamino-2-phenylindole) solution was then added to the DNA
solution to stain the DNA. The final concentrations of DNA and DAPI in the
sample were
4.0 × 10−13and
4.3 × 10−7 M,
respectively. Fluorescence from the stained DNA was measured with
λex = 330–385 nm
and λem ≧420 nm. All solutions
were prepared in Milli Q water. Reagents of analytical grade were used as
received.
8
Single DNA Molecule Observation
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T4 DNA molecules (166 kbp, T4GT7 DNA, Nippon Gene Co., Ltd.) and 7 kbp DNA (Thermo Fisher Scientific Inc.) were used and stained with YOYO-1 at a molar ratio of 1:5 to the total number of base pairs in each sample. For single DNA molecule observations using a microscope, DNA samples were diluted to 5 ng mL -1 with TE buffer (TE (pH 8.0), Nippon Gene Co., Ltd.) and stored in a freezer before use.
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