Restriction Enzymes Sau96I

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 2 matching solutions for this experiment

Sau96I NEB#R0165

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Cells were washed, pelleted, and resuspended in swelling buffer (10 mM Tris pH8.0, 85 mM KCl, 0.5% NP-40, 10 mM MgCl2) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. (For testing whether two REs might increase coverage, in one experiment 100 units of Sau96I and 50 units of DdeI were used in digestion).	Digestion was terminated by adding 40 μl of 10% SDS and 20 μl of 0.5 M EDTA and the chromatin was treated with proteinase K (Ambion) overnight at 55°C. Digested DNA was purified using phenol/chloroform/isoamyl alcohol extractions and precipitated at -80°C for 1 hour. Digested DNA samples were end-repaired and A-tailed as described [63], and ligated with biotinylated and barcoded adaptors.

Protocol tips
Cells were washed, pelleted, and resuspended in swelling buffer (10 mM Tris pH8.0, 85 mM KCl, 0.5% NP-40, 10 mM MgCl2) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. (For testing whether two REs might increase coverage, in one experiment 100 units of Sau96I and 50 units of DdeI were used in digestion).
Downstream tips
Digestion was terminated by adding 40 μl of 10% SDS and 20 μl of 0.5 M EDTA and the chromatin was treated with proteinase K (Ambion) overnight at 55°C. Digested DNA was purified using phenol/chloroform/isoamyl alcohol extractions and precipitated at -80°C for 1 hour. Digested DNA samples were end-repaired and A-tailed as described [63], and ligated with biotinylated and barcoded adaptors.

Publication protocol 1 Relevant paper

Cfr13I (Sau96I) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The URA5-RFLP technique was conducted according to Meyer et al. (2003) PCR products were double digested with Cfr13I (Sau96I) and HhaI enzymes (Thermo Fisher Scientific, Waltham, MA, USA) for 16 h and separated in 3% agarose gel at 100 V for 3 h.

Protocol tips
The URA5-RFLP technique was conducted according to Meyer et al. (2003) PCR products were double digested with Cfr13I (Sau96I) and HhaI enzymes (Thermo Fisher Scientific, Waltham, MA, USA) for 16 h and separated in 3% agarose gel at 100 V for 3 h.

Manufacturer protocol Publication protocol 1 Relevant paper

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