Restriction Enzymes NsiI / Mph1103I

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 3 matching solutions for this experiment

NsiI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Next, the L1 and L2 oligonucleotides of the branched PCR product, which did not participate in the amplification reaction, were annealed in an equimolar ratio to the O1-Comp and O2-Comp oligonucleotides, respectively (Step a3 in Supplementary Fig. 1). This reaction took place at a final concentration of ~270 nM in each strand for 1 h at room temperature in 1× CutSmart buffer (New England Biolabs). This assembly was subsequently combined with the leash precursor at a ~130 nM final concentration, each in 1× CutSmart buffer, along with 10 units (U) of SbfI-HF, 10 U of MluI-HF, 10 U of NsiI-HF, 5 U of AscI, 1,000 U of T4 DNA ligase (New England Biolabs), 1 mM ATP and 1 mM DTT (Step a4 in Supplementary Fig. 1). After overnight incubation at 25 °C, the mixture was heat inactivated at 65 °C for 20 min. Then, in a one-pot reaction that took place at 37 °C for 8–16 h, the tips were nicked with 30 U of Nb.BvCI (New England Biolabs) and the extremities of the shanks were digested with 100 U of XbaI and 50 U of SacI (New England Biolabs) (Step a5 in Supplementary Fig. 1).	The product was separated on a 1.5% agarose gel at 37 °C and extracted using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).

Protocol tips
Next, the L1 and L2 oligonucleotides of the branched PCR product, which did not participate in the amplification reaction, were annealed in an equimolar ratio to the O1-Comp and O2-Comp oligonucleotides, respectively (Step a3 in Supplementary Fig. 1). This reaction took place at a final concentration of ~270 nM in each strand for 1 h at room temperature in 1× CutSmart buffer (New England Biolabs). This assembly was subsequently combined with the leash precursor at a ~130 nM final concentration, each in 1× CutSmart buffer, along with 10 units (U) of SbfI-HF, 10 U of MluI-HF, 10 U of NsiI-HF, 5 U of AscI, 1,000 U of T4 DNA ligase (New England Biolabs), 1 mM ATP and 1 mM DTT (Step a4 in Supplementary Fig. 1). After overnight incubation at 25 °C, the mixture was heat inactivated at 65 °C for 20 min. Then, in a one-pot reaction that took place at 37 °C for 8–16 h, the tips were nicked with 30 U of Nb.BvCI (New England Biolabs) and the extremities of the shanks were digested with 100 U of XbaI and 50 U of SacI (New England Biolabs) (Step a5 in Supplementary Fig. 1).
Downstream tips
The product was separated on a 1.5% agarose gel at 37 °C and extracted using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).

Publication protocol 1 Relevant paper

FastDigest Mph1103I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The resulting plasmid was cut with enzymes Mph1130I and NheI in order to ligate the gene into a Mph1130I‐ and NheI‐digested pNivC vector (with ampicillin resistance marker), where it was attached to ribosome binding site (RBS) ‘C' (AAGTTAAGAGGCAAGA) which allows a moderate translation rate [31].

Protocol tips
The resulting plasmid was cut with enzymes Mph1130I and NheI in order to ligate the gene into a Mph1130I‐ and NheI‐digested pNivC vector (with ampicillin resistance marker), where it was attached to ribosome binding site (RBS) ‘C' (AAGTTAAGAGGCAAGA) which allows a moderate translation rate [31].

Manufacturer protocol Publication protocol 1 Relevant paper

NsiI NEB#R0127

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	RADseq libraries were prepared from extracted DNA according to the method described by Etter et al [22, 23] using the enzymes SbfI-HF and NsiI (New England Biolabs), with 1μg of DNA starting material.

Protocol tips
RADseq libraries were prepared from extracted DNA according to the method described by Etter et al [22, 23] using the enzymes SbfI-HF and NsiI (New England Biolabs), with 1μg of DNA starting material.

Publication protocol 1 Relevant paper

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