Restriction Enzymes AfeI / Eco47III / Aor51HI

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 3 matching solutions for this experiment

Eco47III (AfeI) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	To identify SNPs rs1726866 C/T and rs10246939 G/A, we subjected the 194 bp fragment 2Fmut/2Rmut at two different enzymatic digestions in the presence of endonuclease Eco47III and RsaI, respectively. A fragment modified in that manner shows the sequence AGCGCT for Eco47III and the sequence GTAC for RsaI. A 5 μL aliquot of the 194 bp PCR reaction fragment of was then mixed with a 15 μL solution containing 2 μL 10× buffer O (50 mM Tris-HCl,10 mM MgCl2, 100 mM NaCl, and 0.1 mg/mL BSA pH7.5), 2 μL Eco47III (10 U/mL) (ThermoFisher Scientific,Waltham, MA, USA), and 11 μL sterile deionized H2O. Similarly, a 5 μL aliquot of the same PCR reaction fragment was mixed with a 15 μL solution containing 2 μL 10× Tango buffer (33 mM Tris-acetate, 10 mM Mgacetate, 66 mM K-acetate, and 0.1 mg/mL BSA; pH 7.9), 2 μL RsaI (10 U/mL) (ThermoFisher Scientific), and 11 μL sterile deionized H2O. Each reaction (Eco47III and RsaI) reaction mixture was mixed and then incubated at 37°C for 2 h (Figure 3). The digest (10 μL) was mixed with 3 μL of loading buffer and electrophoresed on a 10% vertical polyacrylamide gel. Silver nitrate and ethidium bromide staining were carried out according to the methods of Herring et al. (1982) and Sambrook et al. (1989), respectively.

Protocol tips
To identify SNPs rs1726866 C/T and rs10246939 G/A, we subjected the 194 bp fragment 2Fmut/2Rmut at two different enzymatic digestions in the presence of endonuclease Eco47III and RsaI, respectively. A fragment modified in that manner shows the sequence AGCGCT for Eco47III and the sequence GTAC for RsaI. A 5 μL aliquot of the 194 bp PCR reaction fragment of was then mixed with a 15 μL solution containing 2 μL 10× buffer O (50 mM Tris-HCl,10 mM MgCl2, 100 mM NaCl, and 0.1 mg/mL BSA pH7.5), 2 μL Eco47III (10 U/mL) (ThermoFisher Scientific,Waltham, MA, USA), and 11 μL sterile deionized H2O. Similarly, a 5 μL aliquot of the same PCR reaction fragment was mixed with a 15 μL solution containing 2 μL 10× Tango buffer (33 mM Tris-acetate, 10 mM Mgacetate, 66 mM K-acetate, and 0.1 mg/mL BSA; pH 7.9), 2 μL RsaI (10 U/mL) (ThermoFisher Scientific), and 11 μL sterile deionized H2O. Each reaction (Eco47III and RsaI) reaction mixture was mixed and then incubated at 37°C for 2 h (Figure 3). The digest (10 μL) was mixed with 3 μL of loading buffer and electrophoresed on a 10% vertical polyacrylamide gel. Silver nitrate and ethidium bromide staining were carried out according to the methods of Herring et al. (1982) and Sambrook et al. (1989), respectively.

Protocol tips

To identify SNPs rs1726866 C/T and rs10246939 G/A, we subjected the 194 bp fragment 2Fmut/2Rmut at two different enzymatic digestions in the presence of endonuclease Eco47III and RsaI, respectively. A fragment modified in that manner shows the sequence AGCGCT for Eco47III and the sequence GTAC for RsaI. A 5 μL aliquot of the 194 bp PCR reaction fragment of was then mixed with a 15 μL solution containing 2 μL 10× buffer O (50 mM Tris-HCl,10 mM MgCl2, 100 mM NaCl, and 0.1 mg/mL BSA pH7.5), 2 μL Eco47III (10 U/mL) (ThermoFisher Scientific,Waltham, MA, USA), and 11 μL sterile deionized H2O. Similarly, a 5 μL aliquot of the same PCR reaction fragment was mixed with a 15 μL solution containing 2 μL
10× Tango buffer (33 mM Tris-acetate, 10 mM Mgacetate, 66 mM K-acetate, and 0.1 mg/mL BSA; pH 7.9), 2 μL RsaI (10 U/mL) (ThermoFisher Scientific), and 11 μL sterile deionized H2O. Each reaction (Eco47III and RsaI) reaction mixture was mixed and then incubated at 37°C for 2 h (Figure 3). The digest (10 μL) was mixed with 3 μL of loading buffer and electrophoresed on a 10% vertical polyacrylamide gel. Silver nitrate and
ethidium bromide staining were carried out according to the methods of Herring et al. (1982) and Sambrook et al. (1989), respectively.

Manufacturer protocol Publication protocol 1 Relevant paper

AfeI NEB#R0652

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Reactions were quenched with 25–30 mM EDTA except in Fig. 2 and Supplemental Fig. S2, where after 90 min the reaction mix was diluted 4-fold in 25 mM Hepes–KOH (pH 7.6), 10 mM Mg(OAc)2, and 60 nM RPA before the addition of water (negative control), PmeI, AvrII, or AfeI (NEB) (3 μl enzyme in 56 μl diluted reaction mix) and incubation at 37 °C for 10 min, followed by quenching with EDTA. Following quenching, proteins were removed with 0.1% SDS and 1/100 volumes proteinase K (NEB P8107) at 37 °C for 20 min, and the DNA was extracted with phenol–chloroform. Samples were passed over illustra MicroSpin G-50 columns (GE Healthcare). Where indicated in figures, samples for the denaturing gel were digested with SmaI. Samples were resolved in native and alkaline (denaturing) agarose gels and visualized by autoradiography or phosphorimaging as described in detail previously [3].

Protocol tips
Reactions were quenched with 25–30 mM EDTA except in Fig. 2 and Supplemental Fig. S2, where after 90 min the reaction mix was diluted 4-fold in 25 mM Hepes–KOH (pH 7.6), 10 mM Mg(OAc)2, and 60 nM RPA before the addition of water (negative control), PmeI, AvrII, or AfeI (NEB) (3 μl enzyme in 56 μl diluted reaction mix) and incubation at 37 °C for 10 min, followed by quenching with EDTA. Following quenching, proteins were removed with 0.1% SDS and 1/100 volumes proteinase K (NEB P8107) at 37 °C for 20 min, and the DNA was extracted with phenol–chloroform. Samples were passed over illustra MicroSpin G-50 columns (GE Healthcare). Where indicated in figures, samples for the denaturing gel were digested with SmaI. Samples were resolved in native and alkaline (denaturing) agarose gels and visualized by autoradiography or phosphorimaging as described in detail previously [3].

Protocol tips

Reactions were quenched with 25–30 mM EDTA except in Fig. 2 and Supplemental Fig. S2, where after 90 min the reaction mix was diluted 4-fold in 25 mM Hepes–KOH (pH 7.6), 10 mM Mg(OAc)2, and 60 nM RPA before the addition of water (negative control), PmeI, AvrII, or AfeI (NEB) (3 μl enzyme in 56 μl diluted reaction mix) and incubation at 37 °C for 10 min, followed by quenching with EDTA.

Following quenching, proteins were removed with 0.1% SDS and 1/100 volumes proteinase K (NEB P8107) at 37 °C for 20 min, and the DNA was extracted with phenol–chloroform. Samples were passed over illustra MicroSpin G-50 columns (GE Healthcare). Where indicated in figures, samples for the denaturing gel were digested with SmaI. Samples were resolved in native and alkaline (denaturing) agarose gels and visualized by autoradiography or phosphorimaging as described in detail previously [3].

Publication protocol 1 Relevant paper

Aor51HI (Eco47III) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Both PCR products were treated with SacI and Aor51HI, and simultaneously cloned into the SacI site of the pFA6a-hphMX6 vector to obtain pSTk14.

Protocol tips
Both PCR products were treated with SacI and Aor51HI, and simultaneously cloned into the SacI site of the pFA6a-hphMX6 vector to obtain pSTk14.

Manufacturer protocol Publication protocol 1 Relevant paper

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