NruI-HF®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The NruI-HF (High Fidelity) Restriction Enzyme (New England Biolabs) was used to linearize the insert, following the manufacturer’s protocol. The digestion mix (50 µL) was prepared with: 1 µL (10 U) of NruI-HF, 1 µg of pIRESneo + ORF A104R, 5 µL of 10× NEBuffer and water, incubated at 37 °C (optimal temperature for NruI), for 60 min.	To purify the construct, a non-toxic method based on ethanol precipitation was used, after restriction enzyme digestion. The volume of DNA was adjusted to 200 µL by adding sterile ddH2O plus 20 µL of 3 M sodium acetate (1/10 of the volume). Then, 2 volumes of 100% ethanol at –20 °C were added to the solution, vortexed for 10 s and placed on a –70 °C freezer for 20 min, subsequently centrifuged for 5 min and the ethanol supernatant was discarded, allowing the pellet to dry. DNA was resuspended in 20 µL of laboratory-grade water.

Protocol tips
The NruI-HF (High Fidelity) Restriction Enzyme (New England Biolabs) was used to linearize the insert, following the manufacturer’s protocol. The digestion mix (50 µL) was prepared with: 1 µL (10 U) of NruI-HF, 1 µg of pIRESneo + ORF A104R, 5 µL of 10× NEBuffer and water, incubated at 37 °C (optimal temperature for NruI), for 60 min.
Downstream tips
To purify the construct, a non-toxic method based on ethanol precipitation was used, after restriction enzyme digestion. The volume of DNA was adjusted to 200 µL by adding sterile ddH2O plus 20 µL of 3 M sodium acetate (1/10 of the volume). Then, 2 volumes of 100% ethanol at –20 °C were added to the solution, vortexed for 10 s and placed on a –70 °C freezer for 20 min, subsequently centrifuged for 5 min and the ethanol supernatant was discarded, allowing the pellet to dry. DNA was resuspended in 20 µL of laboratory-grade water.

Publication protocol

For the extraction of the construct from the E. coli DH5α cells, the EndoFree Plasmid Maxi Kit (Qiagen) was used. E. coli DH5α cultures were grown overnight on LB medium at 37 °C and harvested through centrifugation at 6000× g according to the Qiagen protocol. The NruI-HF (High Fidelity) Restriction Enzyme (New England Biolabs) was used to linearize the insert, following the manufacturer’s protocol. The digestion mix (50 µL) was prepared with: 1 µL (10 U) of NruI-HF, 1 µg of pIRESneo + ORF A104R, 5 µL of 10× NEBuffer and water, incubated at 37 °C (optimal temperature for NruI), for 60 min. To purify the construct, a non-toxic method based on ethanol precipitation was used, after restriction enzyme digestion. The volume of DNA was adjusted to 200 µL by adding sterile ddH2O plus 20 µL of 3 M sodium acetate (1/10 of the volume). Then, 2 volumes of 100% ethanol at –20 °C were added to the solution, vortexed for 10 s and placed on a –70 °C freezer for 20 min, subsequently centrifuged for 5 min and the ethanol supernatant was discarded, allowing the pellet to dry. DNA was resuspended in 20 µL of laboratory-grade water.

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Manufacturer protocol

Download the product protocol from New England BioLabs for NruI-HF® below.

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