Restriction Enzymes Eco52I / EagI

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

Eco52I (EagI) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The purified DNA sample was treated in a 2-step reaction with a set of MREs in cocktail as RE1, including Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI (BssHII)(FastDigest enzyme from Thermal Scientific Inc.), called 4E collectively. Step 1 is the treatment with Cfr42I and PdiI in 1 × Tango Buffer at 37°C for 1 h. Step 2 is the treatment with Eco52I and Ptel after adjusting the buffer to 2 × Tango Buffer and incubating at 37° C for 1 h. Afterward, these MREs were heat-deactivated at 70°C for 10 min. The digested DNA sample obtained was immediately amplified using REPLI-g UltraFast Mini Kit (Qiagen) following the manufacturer-recommended protocol except that denature and neutralization steps were skipped.

Protocol tips
The purified DNA sample was treated in a 2-step reaction with a set of MREs in cocktail as RE1, including Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI (BssHII)(FastDigest enzyme from Thermal Scientific Inc.), called 4E collectively. Step 1 is the treatment with Cfr42I and PdiI in 1 × Tango Buffer at 37°C for 1 h. Step 2 is the treatment with Eco52I and Ptel after adjusting the buffer to 2 × Tango Buffer and incubating at 37° C for 1 h. Afterward, these MREs were heat-deactivated at 70°C for 10 min. The digested DNA sample obtained was immediately amplified using REPLI-g UltraFast Mini Kit (Qiagen) following the manufacturer-recommended protocol except that denature and neutralization steps were skipped.

Protocol tips

The purified DNA sample was treated in a 2-step reaction with a set of MREs in cocktail as RE1, including Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI (BssHII)(FastDigest enzyme from Thermal Scientific Inc.), called 4E collectively. Step 1 is the treatment with Cfr42I and PdiI in 1 × Tango Buffer at 37°C for 1 h. Step 2 is the treatment with Eco52I and Ptel after adjusting the buffer to 2 × Tango Buffer and incubating at 37° C for 1 h. Afterward, these MREs were heat-deactivated at 70°C for 10 min. The digested DNA sample obtained was immediately amplified using REPLI-g UltraFast Mini Kit (Qiagen) following the manufacturer-recommended protocol except that denature and neutralization steps were skipped.

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest Eco52I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	FastDigest® Restriction Enzymes (EagI, EcoRI, FspI, and HindIII) were used according to manufacturer instructions to cleave genomic DNA of the plant materials at specific target nucleotide sequences into DNA fragments of different size. The extracted DNA was subjected to RFLP analysis (Parani et al., 1997). Briefly, the reaction mixture was prepared by adding 10 mL extracted DNA, 15 mL 2X assay buffer, 10 mL bovine serum albumin (BSA), and 3 mL of each restriction enzyme. The vials were incubated at 37°C for 1 h for complete digestion. The restriction enzyme-digested products were visualized through silver staining of the polyacrylamide gel.

Protocol tips
FastDigest® Restriction Enzymes (EagI, EcoRI, FspI, and HindIII) were used according to manufacturer instructions to cleave genomic DNA of the plant materials at specific target nucleotide sequences into DNA fragments of different size. The extracted DNA was subjected to RFLP analysis (Parani et al., 1997). Briefly, the reaction mixture was prepared by adding 10 mL extracted DNA, 15 mL 2X assay buffer, 10 mL bovine serum albumin (BSA), and 3 mL of each restriction enzyme. The vials were incubated at 37°C for 1 h for complete digestion. The restriction enzyme-digested products were visualized through silver staining of the polyacrylamide gel.

Protocol tips

FastDigest® Restriction Enzymes (EagI, EcoRI, FspI, and HindIII) were used according to manufacturer instructions to cleave genomic DNA of the plant materials at specific
target nucleotide sequences into DNA fragments of different size. The extracted DNA was
subjected to RFLP analysis (Parani et al., 1997). Briefly, the reaction mixture was prepared
by adding 10 mL extracted DNA, 15 mL 2X assay buffer, 10 mL bovine serum albumin (BSA),
and 3 mL of each restriction enzyme. The vials were incubated at 37°C for 1 h for complete
digestion. The restriction enzyme-digested products were visualized through silver staining of
the polyacrylamide gel.

Manufacturer protocol Publication protocol 1 Relevant paper

EagI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The thereby assembled complementation cassette was gel-purified, verified by sequencing and further cleaved by EagI-HF (NEB) and SalI-HF (NEB) following manual’s instructions. Analogously, respective pENTRY-miniCR-constructs (see above) were cleaved by the same enzymes, respectively, as a EagI and a SalI restriction site are sequentially located 5 bp upstream of the truncated leader promoter (cf. Fig. 2a).

Protocol tips
The thereby assembled complementation cassette was gel-purified, verified by sequencing and further cleaved by EagI-HF (NEB) and SalI-HF (NEB) following manual’s instructions. Analogously, respective pENTRY-miniCR-constructs (see above) were cleaved by the same enzymes, respectively, as a EagI and a SalI restriction site are sequentially located 5 bp upstream of the truncated leader promoter (cf. Fig. 2a).

Protocol tips

The thereby assembled complementation cassette was gel-purified, verified by sequencing and further cleaved by EagI-HF (NEB) and SalI-HF (NEB) following manual’s instructions. Analogously, respective pENTRY-miniCR-constructs (see above) were cleaved by the same enzymes, respectively, as a EagI and a SalI restriction site are sequentially located 5 bp upstream of the truncated leader promoter (cf. Fig. 2a).

Publication protocol 1 Relevant paper

Eco52I (XmaIII) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	To further confirm LAMP assay results, the amplified products were digested with restriction enzymes and the fragment sizes analyzed by electrophoresis (Eco52I for Beijing genotype strain amplicons) (Fig. 2). Briefly, one microliter of LAMP products was digested overnight by 10 U of Eco52I (Takara, Shiga, Japan) at 37°C and the digested products analyzed by 2% agarose gel electrophoresis.

Protocol tips
To further confirm LAMP assay results, the amplified products were digested with restriction enzymes and the fragment sizes analyzed by electrophoresis (Eco52I for Beijing genotype strain amplicons) (Fig. 2). Briefly, one microliter of LAMP products was digested overnight by 10 U of Eco52I (Takara, Shiga, Japan) at 37°C and the digested products analyzed by 2% agarose gel electrophoresis.

Manufacturer protocol Publication protocol 1 Relevant paper

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