Restriction Enzymes BglI

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 4 matching solutions for this experiment

BglI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	PCR-RFLP method was used to detect Single Nucleotide Polymorphism (SNP) of ORF 38 and 54. The PstI and BglI restriction enzymes (Thermo Fisher Scientific Inc, Waltham, MA USA) was used for digestion reactions with the following protocol: 18 µl Nuclease-free water, 10 µl of PCR product, 2 µl of 10X endonuclease buffer 0, and 1 µl digestion enzymes PstI or BglI. Heating prepared at 37 ⁰C for 15 h. Visualization performed by 2% Agarose gel electrophoresis.

Protocol tips
PCR-RFLP method was used to detect Single Nucleotide Polymorphism (SNP) of ORF 38 and 54. The PstI and BglI restriction enzymes (Thermo Fisher Scientific Inc, Waltham, MA USA) was used for digestion reactions with the following protocol: 18 µl Nuclease-free water, 10 µl of PCR product, 2 µl of 10X endonuclease buffer 0, and 1 µl digestion enzymes PstI or BglI. Heating prepared at 37 ⁰C for 15 h. Visualization performed by 2% Agarose gel electrophoresis.

Protocol tips

PCR-RFLP method was used to detect Single Nucleotide Polymorphism (SNP) of ORF 38 and 54. The PstI and BglI restriction enzymes (Thermo Fisher Scientific Inc, Waltham, MA USA) was used for digestion reactions with the following protocol: 18 µl Nuclease-free water, 10 µl of PCR product, 2 µl of 10X endonuclease buffer 0, and 1 µl digestion enzymes PstI or BglI. Heating prepared at 37 ⁰C for 15 h. Visualization performed by 2% Agarose gel electrophoresis.

Manufacturer protocol Publication protocol 1 Relevant paper

BglI NEB#R0143S

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Using the dimerization of the 30 repeat fragment of ELP1 in the second round of PRe-RDL as an example, the designated ‘A’ fragment was obtained by digestion of 4 μg of ELP1-30 with 10 U AcuI and 40 U BglI for 3 hours at 37°C (see Figure 2A) in NEB Buffer 2 (New England Biolabs; Ipswich, MA). The ‘B’ fragment was obtained by digestion of 4 μg of ELP1-30 with 8 U BseRI and 40 U BglI for 3 hours at 37°C in NEB Buffer 2.

Protocol tips
Using the dimerization of the 30 repeat fragment of ELP1 in the second round of PRe-RDL as an example, the designated ‘A’ fragment was obtained by digestion of 4 μg of ELP1-30 with 10 U AcuI and 40 U BglI for 3 hours at 37°C (see Figure 2A) in NEB Buffer 2 (New England Biolabs; Ipswich, MA). The ‘B’ fragment was obtained by digestion of 4 μg of ELP1-30 with 8 U BseRI and 40 U BglI for 3 hours at 37°C in NEB Buffer 2.

Protocol tips

Using the dimerization of the 30 repeat fragment of ELP1 in the second round of PRe-RDL as an example, the designated ‘A’ fragment was obtained by digestion of 4 μg of ELP1-30 with 10 U AcuI and 40 U BglI for 3 hours at 37°C (see Figure 2A) in NEB Buffer 2 (New England Biolabs; Ipswich, MA). The ‘B’ fragment was obtained by digestion of 4 μg of ELP1-30 with 8 U BseRI and 40 U BglI for 3 hours at 37°C in NEB Buffer 2.

Publication protocol 1 Relevant paper

BglI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Subclone A and subclone G were first digested with SacII and AscI (New England BioLabs), respectively, followed by treatment with calf intestinal alkaline phosphatase (CIAP) (TaKaRa), chloroform extraction, and isopropanol precipitation, and then restricted with BglI (TaKaRa). Subclones B to F were digested with BglI.

Protocol tips
Subclone A and subclone G were first digested with SacII and AscI (New England BioLabs), respectively, followed by treatment with calf intestinal alkaline phosphatase (CIAP) (TaKaRa), chloroform extraction, and isopropanol precipitation, and then restricted with BglI (TaKaRa). Subclones B to F were digested with BglI.

Manufacturer protocol Publication protocol 1 Relevant paper

BglI R6071

Promega

Upstream tips	Protocol tips	Downstream tips
	PCR products were digested with BglI (Promega) for 2 h at 37°C. BglI digestion yields 131, 103 and 60 bp fragments in patients homozygous for the G allele, 163 and 131 bp fragments in patients homozygous for the C allele, and all four fragments in heterozygotes.

Protocol tips
PCR products were digested with BglI (Promega) for 2 h at 37°C. BglI digestion yields 131, 103 and 60 bp fragments in patients homozygous for the G allele, 163 and 131 bp fragments in patients homozygous for the C allele, and all four fragments in heterozygotes.

Manufacturer protocol Publication protocol 1 Relevant paper

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