Restriction Enzymes AvaII / Eco47I / VpaK11BI

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.

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Found 4 matching solutions for this experiment

FastDigest Eco47I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	We first digested the DNA with FastDigest Eco47I restriction enzyme (Thermo Scientific). The final reaction contained 33 ng digested DNA, 900 nM of each primer, and 250 nM of each probe. Twenty microliters of the reaction was loaded into a droplet generator cartridge. Droplets were generated following the manufacturer’s suggested protocol. Cycling conditions were 95° for 10 min, followed by 40 cycles of 94° for 30 sec and 60° for 1 min, and a final 10 min at 98°.

Protocol tips
We first digested the DNA with FastDigest Eco47I restriction enzyme (Thermo Scientific). The final reaction contained 33 ng digested DNA, 900 nM of each primer, and 250 nM of each probe. Twenty microliters of the reaction was loaded into a droplet generator cartridge. Droplets were generated following the manufacturer’s suggested protocol. Cycling conditions were 95° for 10 min, followed by 40 cycles of 94° for 30 sec and 60° for 1 min, and a final 10 min at 98°.

Protocol tips

We first digested the DNA with FastDigest Eco47I restriction enzyme (Thermo Scientific). The final reaction contained 33 ng digested DNA, 900 nM of each primer, and 250 nM of each probe. Twenty microliters of the reaction was loaded into a droplet generator cartridge. Droplets were generated following the manufacturer’s suggested protocol. Cycling conditions were 95° for 10 min, followed by 40 cycles of 94° for 30 sec and 60° for 1 min, and a final 10 min at 98°.

Manufacturer protocol Publication protocol 1 Relevant paper

Eco47I (AvaII) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	PCR products were submitted to enzymatic digestion with Eco47I (Fermentas, Lituania) in the following reaction conditions for a final volume of 15 µL: 1 unit of enzyme (0.1 µL), 1.5 µL of enzyme buffer, 7 µL of PCR product and sterile deionized water. Reactions were incubated at 37 °C for 12 h Digestion products were revealed by 2% agarose gel electrophoresis, subsequently stained with ethidium bromide and visualized using an UV transilluminator.

Protocol tips
PCR products were submitted to enzymatic digestion with Eco47I (Fermentas, Lituania) in the following reaction conditions for a final volume of 15 µL: 1 unit of enzyme (0.1 µL), 1.5 µL of enzyme buffer, 7 µL of PCR product and sterile deionized water. Reactions were incubated at 37 °C for 12 h Digestion products were revealed by 2% agarose gel electrophoresis, subsequently stained with ethidium bromide and visualized using an UV transilluminator.

Protocol tips

PCR products were submitted to enzymatic digestion with Eco47I (Fermentas, Lituania) in the following reaction conditions for a final volume of 15 µL: 1 unit of enzyme (0.1 µL), 1.5 µL of enzyme buffer, 7 µL of PCR product and sterile deionized water. Reactions were incubated at 37 °C for 12 h Digestion products were revealed by 2% agarose gel electrophoresis, subsequently stained with ethidium bromide and visualized using an UV transilluminator.

Manufacturer protocol Publication protocol 1 Relevant paper

VpaK11BI (AvaII) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	After completing duplex PCR, PRA using 2 restriction enzymes, AvaII (TaKaRa) and HaeIII (TaKaRa), was performed to identify the 105 NTM strains that produced 515-bp hsp65 DNA. PRA reactions were performed as follows. Ten microliters of PCR products was transferred to a fresh microcentrifuge tube and digested with restriction enzymes according to the manufacturer's instructions. Following digestion, mixtures were electrophoresed on 3% agarose gel gels.

Protocol tips
After completing duplex PCR, PRA using 2 restriction enzymes, AvaII (TaKaRa) and HaeIII (TaKaRa), was performed to identify the 105 NTM strains that produced 515-bp hsp65 DNA. PRA reactions were performed as follows. Ten microliters of PCR products was transferred to a fresh microcentrifuge tube and digested with restriction enzymes according to the manufacturer's instructions. Following digestion, mixtures were electrophoresed on 3% agarose gel gels.

Protocol tips

After completing duplex PCR, PRA using 2 restriction enzymes, AvaII (TaKaRa) and HaeIII (TaKaRa), was performed to identify the 105 NTM strains that produced 515-bp hsp65 DNA. PRA reactions were performed as follows. Ten microliters of PCR products was transferred to a fresh microcentrifuge tube and digested with restriction enzymes according to the manufacturer's instructions. Following digestion, mixtures were electrophoresed on 3% agarose gel gels.

Manufacturer protocol Publication protocol 1 Relevant paper

AvaII NEB#R0153

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Digestion reactions were performed at 37°C for 30 min. Reaction buffer was based on the NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate) in which commercially available restriction enzyme AvaII (NEB) has 100% activity. AvaII was used in the amounts of 100, 10, 1, 0.1 or 0.01 pmol of dimer in 1 μl volume. Results were visualized by native polyacrylamide or agarose gel electrophoresis.

Protocol tips
Digestion reactions were performed at 37°C for 30 min. Reaction buffer was based on the NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate) in which commercially available restriction enzyme AvaII (NEB) has 100% activity. AvaII was used in the amounts of 100, 10, 1, 0.1 or 0.01 pmol of dimer in 1 μl volume. Results were visualized by native polyacrylamide or agarose gel electrophoresis.

Protocol tips

Digestion reactions were performed at 37°C for 30 min. Reaction buffer was based on the NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate) in which commercially available restriction enzyme AvaII (NEB) has 100% activity. AvaII was used in the amounts of 100, 10, 1, 0.1 or 0.01 pmol of dimer in 1 μl volume. Results were visualized by native polyacrylamide or agarose gel electrophoresis.

Publication protocol 1 Relevant paper

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