Restriction Enzymes ApoI / XapI

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

ApoI NEB#R0566

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	AluI digestion was incubated at 37°C for 4 hours whereas ApoI was incubated at 50°C for overnight. In both digestions, heat inactivation for enzymes was given at 80°C/20 minutes. The restriction products were visualized on a 2.5 % agarose gel containing ethidium bromide. A consistent current at 0.75 m for 2.5 hrs were used for all agarose gel electrophoresis experiments to achieve consistency in RFLP fragment sizes.

Protocol tips
AluI digestion was incubated at 37°C for 4 hours whereas ApoI was incubated at 50°C for overnight. In both digestions, heat inactivation for enzymes was given at 80°C/20 minutes. The restriction products were visualized on a 2.5 % agarose gel containing ethidium bromide. A consistent current at 0.75 m for 2.5 hrs were used for all agarose gel electrophoresis experiments to achieve consistency in RFLP fragment sizes.

Protocol tips

AluI digestion was incubated at 37°C for 4 hours whereas ApoI was incubated at 50°C for overnight. In both digestions, heat inactivation for enzymes was given at 80°C/20 minutes. The restriction products were visualized on a 2.5 % agarose gel containing ethidium bromide. A consistent current at 0.75 m for 2.5 hrs were used for all agarose gel electrophoresis experiments to achieve consistency in RFLP fragment sizes.

Publication protocol 1 Relevant paper

ApoI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	High-fidelity (HF) ApoI and PstI restriction enzymes were obtained from New England BioLabs Inc. (Ipswich, Massachusetts USA). The optimization of restriction enzyme digestion (Supplementary Fig. 4) was performed on 500 ng of FLO1 cell line genomic DNA and included optimization of enzyme concentration, library purification procedure, PCR cycle optimization and removal of FFPE artefacts.

Protocol tips
High-fidelity (HF) ApoI and PstI restriction enzymes were obtained from New England BioLabs Inc. (Ipswich, Massachusetts USA). The optimization of restriction enzyme digestion (Supplementary Fig. 4) was performed on 500 ng of FLO1 cell line genomic DNA and included optimization of enzyme concentration, library purification procedure, PCR cycle optimization and removal of FFPE artefacts.

Publication protocol 1 Relevant paper

FastDigest XapI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The amplified product was digested with FastDigest XapI restriction endonuclease according to manufacturer's recommendations (Thermo Fisher Scientific, Lithuania). Allele-specific RFLP products were separated by electrophoresis on a 1–2% agarose gel and 0.5xTris/Borate/EDTA buffer.

Protocol tips
The amplified product was digested with FastDigest XapI restriction endonuclease according to manufacturer's recommendations (Thermo Fisher Scientific, Lithuania). Allele-specific RFLP products were separated by electrophoresis on a 1–2% agarose gel and 0.5xTris/Borate/EDTA buffer.

Manufacturer protocol Publication protocol 1 Relevant paper

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