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Found 5 matching solutions for this experiment
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We developed an endonuclease assay to study conformations of dsDNA on SWCNTs. The scheme is shown in Figure 1b. In a typical experiment, 0.5 mg/L of DNA–SWCNTs were incubated with 20U of BamHI-HF or EcoRV-HF in NEBuffer 4 (1x) in a total volume of 30 uL in 1.5 mL Eppendorf tubes, at 22°C and shaking with 300 rpm. Aliquots supplemented with Triton X-100 were incubated with the desired Triton X-100 concentration for 2 h at 22 °C before mixing with enzyme and buffer.
After 16 h of incubation, the reaction solutions were diluted to 300 uL with ddH2O before being stopped by adding an equal volume of phenol-chloroform-isoamyl alcohol extraction reagent (Sigma-Aldrich, 25:24:1). After centrifugation for 5 min at 16000 g at room temperature, the aqueous phase was collected, and the DNA was precipitated according to literature using ethanol supplemented with glycogen (CarlRoth). The pellet was washed with 70% ethanol and re-suspended in denaturing
gel loading buffer containing 95% formamide. Samples were heat-denatured for 3 min at 95°C and immediately quenched on ice before separation on a denaturing 12% urea-polyacrylamide gel (Urea-PAGE) in 1X Tris-boric acid-EDTA buffer (TBE) at 250 V for 45 min. |
The DNA was stained with SYBR® Gold (Thermo Fisher) for ~25min and visualized on a blue-light gel imager. The amount of DNA was quantified
using ImageJ. All experiments were conducted in triplicate. |
| Protocol tips |
We developed an endonuclease assay to study conformations of dsDNA on SWCNTs. The scheme is shown in Figure 1b. In a typical experiment, 0.5 mg/L of DNA–SWCNTs were incubated with 20U of BamHI-HF or EcoRV-HF in NEBuffer 4 (1x) in a total volume of 30 uL in 1.5 mL Eppendorf tubes, at 22°C and shaking with 300 rpm. Aliquots supplemented with Triton X-100 were incubated with the desired Triton X-100 concentration for 2 h at 22 °C before mixing with enzyme and buffer.
After 16 h of incubation, the reaction solutions were diluted to 300 uL with ddH2O before being stopped by adding an equal volume of phenol-chloroform-isoamyl alcohol extraction reagent (Sigma-Aldrich, 25:24:1). After centrifugation for 5 min at 16000 g at room temperature, the aqueous phase was collected, and the DNA was precipitated according to literature using ethanol supplemented with glycogen (CarlRoth). The pellet was washed with 70% ethanol and re-suspended in denaturing
gel loading buffer containing 95% formamide. Samples were heat-denatured for 3 min at 95°C and immediately quenched on ice before separation on a denaturing 12% urea-polyacrylamide gel (Urea-PAGE) in 1X Tris-boric acid-EDTA buffer (TBE) at 250 V for 45 min. |
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The DNA was stained with SYBR® Gold (Thermo Fisher) for ~25min and visualized on a blue-light gel imager. The amount of DNA was quantified
using ImageJ. All experiments were conducted in triplicate. |
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The test for the presence of cohesive ends of the phage genome was performed as previously described (26), using the restriction enzymes HindIII, XhoI, Eco32I, SalI, and SmaI (Thermo Scientific). Briefly, phage DNA samples were divided into two parts after restriction digestion. The first part was loaded on the gel at 0°C, whereas the second part was heated at 70°C for 10 min prior to loading. Subsequently, all the samples were immediately separated by electrophoresis, and the DNA band patterns were compared. |
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| The test for the presence of cohesive ends of the phage genome was performed as previously described (26), using the restriction enzymes HindIII, XhoI, Eco32I, SalI, and SmaI (Thermo Scientific). Briefly, phage DNA samples were divided into two parts after restriction digestion. The first part was loaded on the gel at 0°C, whereas the second part was heated at 70°C for 10 min prior to loading. Subsequently, all the samples were immediately separated by electrophoresis, and the DNA band patterns were compared. |
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After amplification, the ETAS-PCR/Vgsc-1014 multiplex products were digested with FastDigest Eco32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water. The reaction was incubated at 37 °C for 10 min. Finally, 10 µl of digestion reaction was loaded on a 3% agarose gel. |
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| After amplification, the ETAS-PCR/Vgsc-1014 multiplex products were digested with FastDigest Eco32I restriction enzyme (Thermo Scientific, United Kingdom) in a final reaction volume of 30 µl including 10 µl of the PCR product, 2 µl of 10× FastDigest Green buffer, 1.5 µl of FastDigest enzyme and 17 µl distilled water. The reaction was incubated at 37 °C for 10 min. Finally, 10 µl of digestion reaction was loaded on a 3% agarose gel. |
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Digestion reactions were set up in 12 μl volume using 96 fmoles of duplex per reaction, with SUPERase•In™ RNase Inhibitor 1 U/μl, in the buffer recommended by the manufacturer. Enzymes were used at a maximum concentration compatible with glycerol content lower than 4.75%. MvaI, HindIII, PvuII (Fastdigest™ series) and BcnI enzymes were from Thermo Scientific™. AvaII, HinP1I and EcoRV were from New England Biolabs. |
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| Digestion reactions were set up in 12 μl volume using 96 fmoles of duplex per reaction, with SUPERase•In™ RNase Inhibitor 1 U/μl, in the buffer recommended by the manufacturer. Enzymes were used at a maximum concentration compatible with glycerol content lower than 4.75%. MvaI, HindIII, PvuII (Fastdigest™ series) and BcnI enzymes were from Thermo Scientific™. AvaII, HinP1I and EcoRV were from New England Biolabs. |
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The amplified fragment was first cloned into a pMD-18T vector (Takara Bio Inc.) with fusion green fluorescent protein expression and then subcloned (BamHI + EcoRV; Takara Bio Inc.) into a pWPXL lentivirus vector. |
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| The amplified fragment was first cloned into a pMD-18T vector (Takara Bio Inc.) with fusion green fluorescent protein expression and then subcloned (BamHI + EcoRV; Takara Bio Inc.) into a pWPXL lentivirus vector. |
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