Restriction Enzymes NruI / Bsp68I

A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence. The four most common types of restriction enzymes include: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). The most common challenges with restriction digest include- 1. inactivation of the enzyme, 2. incomplete or no digestion, and 3. unexpected cleavage. The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, the optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with the activity of the other. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.

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Found 4 matching solutions for this experiment

NruI-HF®

New England BioLabs

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 1 Relevant paper

NruI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Intact λDNA (0.3 μg mL−1, 3010, Takara, Shiga, Japan) shown in step 1 of Fig. 2 was first digested by a restriction enzyme, NruI (1168A, Takara), at 37 °C for one hour, providing six fragments (Fig. 2, step 2).	Fragments of 4.6 kbp and 6.7 kbp were purified for labeling with biotin and digoxin, respectively, using NucleoSpin Extract II (740609, Macherey-Nagel, Düren, Germany) after electrophoresis in 0.8% agarose gel. Each fragment was labeled at 37 °C for one hour in separate Eppendorf tubes following the user instructions for the nucleic acid labeling kits used, Label IT® Digoxin Labeling Kit (MIR3325, Mirus, Madison, WI, USA) and Label IT® Biotin Labeling Kit (MIR3425, Mirus), as shown in step 3 of Fig. 2. Fragments were purified from free digoxin or biotin by G50 microspin purification columns (Mirus).

Protocol tips
Intact λDNA (0.3 μg mL−1, 3010, Takara, Shiga, Japan) shown in step 1 of Fig. 2 was first digested by a restriction enzyme, NruI (1168A, Takara), at 37 °C for one hour, providing six fragments (Fig. 2, step 2).
Downstream tips
Fragments of 4.6 kbp and 6.7 kbp were purified for labeling with biotin and digoxin, respectively, using NucleoSpin Extract II (740609, Macherey-Nagel, Düren, Germany) after electrophoresis in 0.8% agarose gel. Each fragment was labeled at 37 °C for one hour in separate Eppendorf tubes following the user instructions for the nucleic acid labeling kits used, Label IT® Digoxin Labeling Kit (MIR3325, Mirus, Madison, WI, USA) and Label IT® Biotin Labeling Kit (MIR3425, Mirus), as shown in step 3 of Fig. 2. Fragments were purified from free digoxin or biotin by G50 microspin purification columns (Mirus).

Manufacturer protocol Publication protocol 1 Relevant paper

Bsp68I (NruI) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Restriction digestion of the 300-bp product, obtained from PCR of all the clinical isolates, the reference strains, and the spiked sputum sample, was carried out with 1 U each of NruI and BamHI (Fermentas Life Sciences, Lithuania) in separate tubes. Restriction digestion was carried out with 10 μl of the amplicon at 37°C for 2 h.	The products were electrophoresed on 2.5% agarose gel with a 50-bp DNA marker (Fermentas Life Sciences, Lithuania).

Protocol tips
Restriction digestion of the 300-bp product, obtained from PCR of all the clinical isolates, the reference strains, and the spiked sputum sample, was carried out with 1 U each of NruI and BamHI (Fermentas Life Sciences, Lithuania) in separate tubes. Restriction digestion was carried out with 10 μl of the amplicon at 37°C for 2 h.
Downstream tips
The products were electrophoresed on 2.5% agarose gel with a 50-bp DNA marker (Fermentas Life Sciences, Lithuania).

Manufacturer protocol Publication protocol 1 Relevant paper

NruI NEB#R0192

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	For direct assembly of transformable plasmids by cycled ligation, a modified pUC19 vector (Table S1) with two blunt ligation sites separated by ∼250 bp was created. The vector was digested with NruI and SwaI (NEB) and the linearized vector was isolated and purified by gel extraction (Qiagen). Detailed protocol in Table S3.

Protocol tips
For direct assembly of transformable plasmids by cycled ligation, a modified pUC19 vector (Table S1) with two blunt ligation sites separated by ∼250 bp was created. The vector was digested with NruI and SwaI (NEB) and the linearized vector was isolated and purified by gel extraction (Qiagen). Detailed protocol in Table S3.

Publication protocol 1 Relevant paper

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