Restriction Enzymes SspI

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 5 matching solutions for this experiment

FastDigest SspI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point.

Protocol tips
Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point.

Protocol tips

Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point.

Manufacturer protocol Publication protocol 1 Relevant paper

SspI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	5 μl of the PCR product and 2 μl of the restriction enzyme were prepared by mixing 2 μl of 10x buffer, and sterile free water was added for a total volume of 20 μl. The tubes were incubated at 37°C for approximately 4 h. The digested PCR products were then separated on 2.5% agarose gel.

Protocol tips
5 μl of the PCR product and 2 μl of the restriction enzyme were prepared by mixing 2 μl of 10x buffer, and sterile free water was added for a total volume of 20 μl. The tubes were incubated at 37°C for approximately 4 h. The digested PCR products were then separated on 2.5% agarose gel.

Manufacturer protocol Publication protocol 1 Relevant paper

SspI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Fluorescently labeled products were digested with 10 U of restriction enzyme SspI or AvrII (Thermo Fisher Scientific, Italy) by following the manufacturer’s instructions.	Digested products were purified and analyzed on an ABI3730 capillary sequencer in genotyping mode with the size standard ROX-labeled GS500.

Protocol tips
Fluorescently labeled products were digested with 10 U of restriction enzyme SspI or AvrII (Thermo Fisher Scientific, Italy) by following the manufacturer’s instructions.
Downstream tips
Digested products were purified and analyzed on an ABI3730 capillary sequencer in genotyping mode with the size standard ROX-labeled GS500.

Manufacturer protocol Publication protocol 1 Relevant paper

SspI NEB#R0132

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	0.5 to 4 μg of Phi X 174 gDNA (Thermo Scientific) was restriction-digested using 5U of SspI (NEB) in 1× SspI Reaction Buffer for 2 hours at 37°C to linearize DNA and produce blunt ends followed by SPRI bead purification.

Protocol tips
0.5 to 4 μg of Phi X 174 gDNA (Thermo Scientific) was restriction-digested using 5U of SspI (NEB) in 1× SspI Reaction Buffer for 2 hours at 37°C to linearize DNA and produce blunt ends followed by SPRI bead purification.

Publication protocol 1 Relevant paper

SspI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

Protocol tips
Twenty micrograms of each DNA sample was digested overnight at 37°C with either AflII-HF or SspI-HF restriction endonucleases according to the manufacturer's recommendations (New England BioLabs, Ipswich, MA).

Publication protocol 1 Relevant paper

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