Restriction Enzymes Bsp1286I / SduI

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 inclue: 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 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, 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 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.

2 Matching solutions
Start a discussion

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 2 matching solutions for this experiment

Bsp1286I (SduI) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	The PCR product (709 bp) was treated with restriction endonuclease Bsp1286I (Takara Biotechnology, Otsu, Japan), size-separated on a 2% agarose gel with ethidium bromide, and visualized with UV light. In HNA-5a-positive homozygote samples, three fragments of 297 bp, 217 bp, and 195 bp were generated; in HNA-5a-negative homozygote samples, two fragments of 412 bp and 297 bp were generated; and in HNA-5a heterozygote samples, four fragments of 412 bp, 297 bp, 217 bp, and 195 bp were generated (Fig. 2).

Protocol tips
The PCR product (709 bp) was treated with restriction endonuclease Bsp1286I (Takara Biotechnology, Otsu, Japan), size-separated on a 2% agarose gel with ethidium bromide, and visualized with UV light. In HNA-5a-positive homozygote samples, three fragments of 297 bp, 217 bp, and 195 bp were generated; in HNA-5a-negative homozygote samples, two fragments of 412 bp and 297 bp were generated; and in HNA-5a heterozygote samples, four fragments of 412 bp, 297 bp, 217 bp, and 195 bp were generated (Fig. 2).

Protocol tips

The PCR product (709 bp) was treated with restriction endonuclease Bsp1286I (Takara Biotechnology, Otsu, Japan), size-separated on a 2% agarose gel with ethidium bromide, and visualized with UV light. In HNA-5a-positive homozygote samples, three fragments of 297 bp, 217 bp, and 195 bp were generated; in HNA-5a-negative homozygote samples, two fragments of 412 bp and 297 bp were generated; and in HNA-5a heterozygote samples, four fragments of 412 bp, 297 bp, 217 bp, and 195 bp were generated (Fig. 2).

Manufacturer protocol Publication protocol 1 Relevant paper

Bsp1286I NEB#R0120

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	For each sample, digestion and ligation reactions were carried out simultaneously at 37°C for 3 hours on 50 ng of genomic DNA, using 2 units of restriction enzyme Bsp1286I (New England Biolabs, NEB), 80 units of T4 DNA ligase (NEB) and 0.05 μM Bsp1286I adaptors I (Table (Table1),1), in a buffer with final concentrations of 10 mM Tris-OAc, 50 mM KOAc, 10 mM Mg(OAc)2, 5 mM DTT (pH 7.8), 1 mM ATP and 100 ng/ml Bovine Serum Albumin (NEB).

Protocol tips
For each sample, digestion and ligation reactions were carried out simultaneously at 37°C for 3 hours on 50 ng of genomic DNA, using 2 units of restriction enzyme Bsp1286I (New England Biolabs, NEB), 80 units of T4 DNA ligase (NEB) and 0.05 μM Bsp1286I adaptors I (Table (Table1),1), in a buffer with final concentrations of 10 mM Tris-OAc, 50 mM KOAc, 10 mM Mg(OAc)2, 5 mM DTT (pH 7.8), 1 mM ATP and 100 ng/ml Bovine Serum Albumin (NEB).

Protocol tips

For each sample, digestion and ligation reactions were carried out simultaneously at 37°C for 3 hours on 50 ng of genomic DNA, using 2 units of restriction enzyme Bsp1286I (New England Biolabs, NEB), 80 units of T4 DNA ligase (NEB) and 0.05 μM Bsp1286I adaptors I (Table (Table1),1), in a buffer with final concentrations of 10 mM Tris-OAc, 50 mM KOAc, 10 mM Mg(OAc)2, 5 mM DTT (pH 7.8), 1 mM ATP and 100 ng/ml Bovine Serum Albumin (NEB).

Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment