Restriction Enzymes SsiI / AciI

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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AciI NEB#R0551

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The PCR products were cut using the following restriction enzymes: fliC was cut with HhaI and Sau3AI; gnd with AciI and AluI; and mutS with AciI and HaeII. Restriction enzymes from New England Biolabs (Ipswich, MA) and Fermentas (Glen Burnie, MD) were used during the development of the molecular typing method. Single digestions were done by mixing 5 μl of the selected PCR product and 2.5U of NEB endonucleases or 1 Fast digest unit of the Fermentas endonucleases in final volume of 10 μl. NEB endonuclease mixtures were incubated for 1 h at 37°C. Fast digest mixtures were incubated for 10 min at 37°C. After incubation, DNA digestion was terminated by heat inactivation at 65°C for 20 or 10 min depending on the enzyme used or by the addition of 20 mM EDTA. In selected experiments, restriction digestions were cleaned using the MinElute Reaction Cleanup kit (Qiagen). Restriction digestions were repeated two to three times to test reproducibility of the restriction patterns. Restriction patterns were analyzed using the Agilent DNA 1000 kit and the 2100 Agilent Bioanalyzer (Agilent Technologies, Inc.).

Protocol tips
The PCR products were cut using the following restriction enzymes: fliC was cut with HhaI and Sau3AI; gnd with AciI and AluI; and mutS with AciI and HaeII. Restriction enzymes from New England Biolabs (Ipswich, MA) and Fermentas (Glen Burnie, MD) were used during the development of the molecular typing method. Single digestions were done by mixing 5 μl of the selected PCR product and 2.5U of NEB endonucleases or 1 Fast digest unit of the Fermentas endonucleases in final volume of 10 μl. NEB endonuclease mixtures were incubated for 1 h at 37°C. Fast digest mixtures were incubated for 10 min at 37°C. After incubation, DNA digestion was terminated by heat inactivation at 65°C for 20 or 10 min depending on the enzyme used or by the addition of 20 mM EDTA. In selected experiments, restriction digestions were cleaned using the MinElute Reaction Cleanup kit (Qiagen). Restriction digestions were repeated two to three times to test reproducibility of the restriction patterns. Restriction patterns were analyzed using the Agilent DNA 1000 kit and the 2100 Agilent Bioanalyzer (Agilent Technologies, Inc.).

Protocol tips

The PCR products were cut using the following restriction enzymes: fliC was cut with HhaI and Sau3AI; gnd with AciI and AluI; and mutS with AciI and HaeII. Restriction enzymes from New England Biolabs (Ipswich, MA) and Fermentas (Glen Burnie, MD) were used during the development of the molecular typing method. Single digestions were done by mixing 5 μl of the selected PCR product and 2.5U of NEB endonucleases or 1 Fast digest unit of the Fermentas endonucleases in final volume of 10 μl. NEB endonuclease mixtures were incubated for 1 h at 37°C. Fast digest mixtures were incubated for 10 min at 37°C. After incubation, DNA digestion was terminated by heat inactivation at 65°C for 20 or 10 min depending on the enzyme used or by the addition of 20 mM EDTA. In selected experiments, restriction digestions were cleaned using the MinElute Reaction Cleanup kit (Qiagen). Restriction digestions were repeated two to three times to test reproducibility of the restriction patterns. Restriction patterns were analyzed using the Agilent DNA 1000 kit and the 2100 Agilent Bioanalyzer (Agilent Technologies, Inc.).

Publication protocol 1 Relevant paper

FastDigest SsiI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	NA digestion reactions were set up with 200 ng DNA and 1 µL of each enzyme (AciI, MspI or HpaII FastDigest - purchased from Fermentas) in a total volume of 20 µL and incubated for 1 h at 37°C. Reaction products were loaded on 2% MetaPhor high resolution agarose (Lonza) gel with 0.4 µg/mL of Ethidium Bromide.

Protocol tips
NA digestion reactions were set up with 200 ng DNA and 1 µL of each enzyme (AciI, MspI or HpaII FastDigest - purchased from Fermentas) in a total volume of 20 µL and incubated for 1 h at 37°C. Reaction products were loaded on 2% MetaPhor high resolution agarose (Lonza) gel with 0.4 µg/mL of Ethidium Bromide.

Manufacturer protocol Publication protocol 1 Relevant paper

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