Restriction Enzymes BbvI / Lsp1109I

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 2 matching solutions for this experiment

BbvI NEB#R0173

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Escherichia coli genomic DNA was sourced from the MG1655 strain supplied from the American Type Culture Collection (Manassas, VA). DNA was isolated by lysis of bacterial cells in an SDS–proteinase K solution followed by RNAse treatment and multiple phenol/chloroform extractions before ethanol precipitation (16). Human genomic DNA was purchased from Promega (Madison, WI). The DNA (10 µg) was digested with 8 U of the restriction enzyme BbvI (NEB) for 3 h at 37°C before heat inactivation at 65°C for 20 min.

Protocol tips
Escherichia coli genomic DNA was sourced from the MG1655 strain supplied from the American Type Culture Collection (Manassas, VA). DNA was isolated by lysis of bacterial cells in an SDS–proteinase K solution followed by RNAse treatment and multiple phenol/chloroform extractions before ethanol precipitation (16). Human genomic DNA was purchased from Promega (Madison, WI). The DNA (10 µg) was digested with 8 U of the restriction enzyme BbvI (NEB) for 3 h at 37°C before heat inactivation at 65°C for 20 min.

Protocol tips

Escherichia coli genomic DNA was sourced from the MG1655 strain supplied from the American Type Culture Collection (Manassas, VA). DNA was isolated by lysis of bacterial cells in an SDS–proteinase K solution followed by RNAse treatment and multiple phenol/chloroform extractions before ethanol precipitation (16). Human genomic DNA was purchased from Promega (Madison, WI). The DNA (10 µg) was digested with 8 U of the restriction enzyme BbvI (NEB) for 3 h at 37°C before heat inactivation at 65°C for 20 min.

Publication protocol 1 Relevant paper

FastDigest Lsp1109I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	PCR products were column-purified using a nucleospin extract II kit (Macherey-Nagel) and recovered in 40 μl H20. Initial biotinylated di- and tri-blocks were digested with SfaNI (FastDigest, Fermentas); terminal di-modules were digested with BbvI (FastDigest Lsp1109I, Fermentas). Digested fragments were column purified and quantified (Nanodrop, Thermo scientific). Two micrograms of purified digested PCR products were typically obtained with this process.

Protocol tips
PCR products were column-purified using a nucleospin extract II kit (Macherey-Nagel) and recovered in 40 μl H20. Initial biotinylated di- and tri-blocks were digested with SfaNI (FastDigest, Fermentas); terminal di-modules were digested with BbvI (FastDigest Lsp1109I, Fermentas). Digested fragments were column purified and quantified (Nanodrop, Thermo scientific). Two micrograms of purified digested PCR products were typically obtained with this process.

Protocol tips

PCR products were column-purified using a nucleospin extract II kit (Macherey-Nagel) and recovered in 40 μl H20. Initial biotinylated di- and tri-blocks were digested with SfaNI (FastDigest, Fermentas); terminal di-modules were digested with BbvI (FastDigest Lsp1109I, Fermentas). Digested fragments were column purified and quantified (Nanodrop, Thermo scientific). Two micrograms of purified digested PCR products were typically obtained with this process.

Manufacturer protocol Publication protocol 1 Relevant paper

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