Restriction Enzymes BstZ17I / Bst1107I

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 2 matching solutions for this experiment

BstZ17I-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The annealed DNA icosahedron samples were treated with different restriction enzyme cocktails for corresponding patterns (BsiWI-HF, BstZ17I-HF, MfeI-HF, MluI-HF for pattern I; AclI, BamHI-HF, MfeI-HF, MluI-HF for pattern II; AclI, BamHI-HF, BsiWI-HF, BstZ17I-HF, MfeI-HF, MluI-HF for pattern III). One unit of each enzyme was used to digest the icosahedron in 50 µL 1 × CutSmart buffer at 37 °C for 2 h. Additional details can be found in Supplementary Methods.

Protocol tips
The annealed DNA icosahedron samples were treated with different restriction enzyme cocktails for corresponding patterns (BsiWI-HF, BstZ17I-HF, MfeI-HF, MluI-HF for pattern I; AclI, BamHI-HF, MfeI-HF, MluI-HF for pattern II; AclI, BamHI-HF, BsiWI-HF, BstZ17I-HF, MfeI-HF, MluI-HF for pattern III). One unit of each enzyme was used to digest the icosahedron in 50 µL 1 × CutSmart buffer at 37 °C for 2 h. Additional details can be found in Supplementary Methods.

Protocol tips

The annealed DNA icosahedron samples were treated with different restriction enzyme cocktails for corresponding patterns (BsiWI-HF, BstZ17I-HF, MfeI-HF, MluI-HF for pattern I; AclI, BamHI-HF, MfeI-HF, MluI-HF for pattern II; AclI, BamHI-HF, BsiWI-HF, BstZ17I-HF, MfeI-HF, MluI-HF for pattern III). One unit of each enzyme was used to digest the icosahedron in 50 µL 1 × CutSmart buffer at 37 °C for 2 h. Additional details can be found in Supplementary Methods.

Publication protocol 1 Relevant paper

FastDigest Bst1107I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Ninety percent confluent cells cultured in a 25 cm2 flask were transfected with 10 μg of linearized vector pSwitch (Bst1107 I restriction endonuclease) using 25 μL of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Protocol tips
Ninety percent confluent cells cultured in a 25 cm2 flask were transfected with 10 μg of linearized vector pSwitch (Bst1107 I restriction endonuclease) using 25 μL of lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

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