EcoRV-HF®

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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.
Downstream tips
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.

Publication protocol

"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 (Carl
Roth).57 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 ~25
min 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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