Restriction Enzymes Bsh1236I / BstUI

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

FastDigest Bsh1236I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1 volumes of 3 M sodium acetate (pH 4.6) at −20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 μL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 μL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). The DNA fragments were size-separated by capillary electrophoresis (CEQ™ 8800; Beckman Coulter). The conditions of the runs corresponded to the Frag-4 programme described by the manufacturer, but the duration of the runs was extended to 80 min.

Protocol tips
Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1 volumes of 3 M sodium acetate (pH 4.6) at −20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 μL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 μL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). The DNA fragments were size-separated by capillary electrophoresis (CEQ™ 8800; Beckman Coulter). The conditions of the runs corresponded to the Frag-4 programme described by the manufacturer, but the duration of the runs was extended to 80 min.

Protocol tips

Three independent fingerprints were generated for Bacteria and Fungi with HaeIII (BsuRI), FastDigest HhaI (both Fermentas/Thermo Fisher Scientific, Waltham), and MspI (New England Biolabs), respectively, while archaeal PCR products were digested only with BstUI (Fermentas/Thermo Fisher Scientific). The incubations were run for 4 h at 37 °C. The reactions were stopped by freezing at -20 °C. The DNA was then precipitated with 5 volumes of 95% (v/v) ethanol and 0.1 volumes of 3 M sodium acetate (pH 4.6) at −20 °C for 30 min. After centrifugation with 14 000 g for 30 min at 4 °C, the DNA pellets were washed with 100 μL ice-cold 70% (v/v) ethanol and centrifuged again for 10 min. The pellets were then air-dried, dissolved in 30 μL sample loading solution and supplied with internal standard 600 (both Beckman Coulter, Brea). The DNA fragments were size-separated by capillary electrophoresis (CEQ™ 8800; Beckman Coulter). The conditions of the runs corresponded to the Frag-4 programme described by the manufacturer, but the duration of the runs was extended to 80 min.

Manufacturer protocol Publication protocol 1 Relevant paper

BstUI NEB#R0518

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Enzyme digestion was conducted in a 20 μL final volume with 5U of BstUI enzyme (New England Biolabs [NEB]) and 10 μL of PCR product. The reaction was incubated at 60℃ overnight. The digested products were visualized on 3% agarose gels stained with ethidium bromide.

Protocol tips
Enzyme digestion was conducted in a 20 μL final volume with 5U of BstUI enzyme (New England Biolabs [NEB]) and 10 μL of PCR product. The reaction was incubated at 60℃ overnight. The digested products were visualized on 3% agarose gels stained with ethidium bromide.

Publication protocol 1 Relevant paper

Bsh1236I (BstUI) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Amplification products obtained by PCR were digested with two restriction enzymes, HaeIII (Sigma) and BstUI (Thermo Scientific), following the manufacturer's recommendations.

Protocol tips
Amplification products obtained by PCR were digested with two restriction enzymes, HaeIII (Sigma) and BstUI (Thermo Scientific), following the manufacturer's recommendations.

Manufacturer protocol Publication protocol 1 Relevant paper

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