Restriction Enzymes DdeI / HpyF3I

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 4 matching solutions for this experiment

DdeI NEB#R0175

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The amplified PCR product of 124 bp length was digested at 37 °C using one unit of DdeI (NEB, Beverly, MA, USA) restriction enzyme. This enzyme recognises the CTG sequence, originating in two fragments of 104 bp and 20 bp length. The wild-type allele (GG) produced one band (124 bp) while the wild type/variant allele (GA) produced three bands of 124 bp, 104 bp and 20 bp. The variant allele (AA) produced two bands of 104 bp and 20 bp length when run on 2.5% agarose gel electrophoresis.

Protocol tips
The amplified PCR product of 124 bp length was digested at 37 °C using one unit of DdeI (NEB, Beverly, MA, USA) restriction enzyme. This enzyme recognises the CTG sequence, originating in two fragments of 104 bp and 20 bp length. The wild-type allele (GG) produced one band (124 bp) while the wild type/variant allele (GA) produced three bands of 124 bp, 104 bp and 20 bp. The variant allele (AA) produced two bands of 104 bp and 20 bp length when run on 2.5% agarose gel electrophoresis.

Protocol tips

The amplified PCR product of 124 bp length was digested at 37 °C using one unit of DdeI (NEB, Beverly, MA, USA) restriction enzyme. This enzyme recognises the CTG sequence, originating in two fragments of 104 bp and 20 bp length. The wild-type allele (GG) produced one band (124 bp) while the wild type/variant allele (GA) produced three bands of 124 bp, 104 bp and 20 bp. The variant allele (AA) produced two bands of 104 bp and 20 bp length when run on 2.5% agarose gel electrophoresis.

Publication protocol 1 Relevant paper

DdeI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	A fragment that included the polymorphic nucleotide position was amplified and digested with endonuclease DdeI or Kpn2I (Takara Bio Inc., Shiga, Japan). Digested DNA fragments were separated on a 2.5% agarose gel and visualized after staining with ethidium bromide.

Protocol tips
A fragment that included the polymorphic nucleotide position was amplified and digested with endonuclease DdeI or Kpn2I (Takara Bio Inc., Shiga, Japan). Digested DNA fragments were separated on a 2.5% agarose gel and visualized after staining with ethidium bromide.

Manufacturer protocol Publication protocol 1 Relevant paper

DdeI R6291

Promega

Upstream tips	Protocol tips	Downstream tips
	Aliquots of 10 μL of each 275 bp amplified product were digested with restriction endonucleases as recommended by the manufacturer. Three restriction enzymes, DdeI, HaeIII and HinfI (Promega, Madison, WI, USA), were used in separate reactions.

Protocol tips
Aliquots of 10 μL of each 275 bp amplified product were digested with restriction endonucleases as recommended by the manufacturer. Three restriction enzymes, DdeI, HaeIII and HinfI (Promega, Madison, WI, USA), were used in separate reactions.

Manufacturer protocol Publication protocol 1 Relevant paper

HpyF3I (DdeI) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	After amplification of the flaA fragment, a 10-μL sample (0.1–0.5 μg of amplified DNA quantified by Nanodrop®, Canada) of the amplicons was digested with the restriction enzyme HpyF3I (Thermo Scientific®) according to the manufacturer’s instructions. Briefly, 10 μL of PCR product, 18 μL nuclease-free water, 2 μL Buffer Tango (10×, Thermo Scientific®), and 1–2 μL of HpyF3I enzyme were added together in 0.2-mL DNase-free tubes and incubated at 37 °C for 16 h. Then, digests were analyzed on a 2.5% agarose gel containing 0.5 μg/mL ethidium bromide. Low-range DNA ladder (50 bp, Jena Bioscience) was used as standard for molecular size determination.

Protocol tips
After amplification of the flaA fragment, a 10-μL sample (0.1–0.5 μg of amplified DNA quantified by Nanodrop®, Canada) of the amplicons was digested with the restriction enzyme HpyF3I (Thermo Scientific®) according to the manufacturer’s instructions. Briefly, 10 μL of PCR product, 18 μL nuclease-free water, 2 μL Buffer Tango (10×, Thermo Scientific®), and 1–2 μL of HpyF3I enzyme were added together in 0.2-mL DNase-free tubes and incubated at 37 °C for 16 h. Then, digests were analyzed on a 2.5% agarose gel containing 0.5 μg/mL ethidium bromide. Low-range DNA ladder (50 bp, Jena Bioscience) was used as standard for molecular size determination.

Protocol tips

After amplification of the flaA fragment, a 10-μL sample (0.1–0.5 μg of amplified DNA quantified by Nanodrop®, Canada) of the amplicons was digested with the restriction enzyme HpyF3I (Thermo Scientific®) according to the manufacturer’s instructions. Briefly, 10 μL of PCR product, 18 μL nuclease-free water, 2 μL Buffer Tango (10×, Thermo Scientific®), and 1–2 μL of HpyF3I enzyme were added together in 0.2-mL DNase-free tubes and incubated at 37 °C for 16 h. Then, digests were analyzed on a 2.5% agarose gel containing 0.5 μg/mL ethidium bromide. Low-range DNA ladder (50 bp, Jena Bioscience) was used as standard for molecular size determination.

Manufacturer protocol Publication protocol 1 Relevant paper

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