Restriction Enzymes ClaI

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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ClaI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Two PCR products containing each mutation were digested by restriction endonucleases as follows. PCR products amplified by primer pair (iii) or (iv) were incubated with FokI (Takara‐Bio Co., Ltd, Kusatsu, Japan), and those amplified by primer pair (iv) were incubated with ClaI (Takara‐Bio Co.) for over 4 h, in order to identify the missense mutation in β3 exon 5 and exon 6. The digested PCR products were analyzed by electrophoresis using a 7% polyacrylamide gel. The PCR products after electrophoresis were visualized by staining with ethidium bromide solution (Wako Pure Chemical Industries).

Protocol tips
Two PCR products containing each mutation were digested by restriction endonucleases as follows. PCR products amplified by primer pair (iii) or (iv) were incubated with FokI (Takara‐Bio Co., Ltd, Kusatsu, Japan), and those amplified by primer pair (iv) were incubated with ClaI (Takara‐Bio Co.) for over 4 h, in order to identify the missense mutation in β3 exon 5 and exon 6. The digested PCR products were analyzed by electrophoresis using a 7% polyacrylamide gel. The PCR products after electrophoresis were visualized by staining with ethidium bromide solution (Wako Pure Chemical Industries).

Protocol tips

Two PCR products containing each mutation were digested by restriction endonucleases as follows. PCR products amplified by primer pair (iii) or (iv) were incubated with FokI (Takara‐Bio Co., Ltd, Kusatsu, Japan), and those amplified by primer pair (iv) were incubated with ClaI (Takara‐Bio Co.) for over 4 h, in order to identify the missense mutation in β3 exon 5 and exon 6. The digested PCR products were analyzed by electrophoresis using a 7% polyacrylamide gel. The PCR products after electrophoresis were visualized by staining with ethidium bromide solution (Wako Pure Chemical Industries).

Manufacturer protocol Publication protocol 1 Relevant paper

ClaI NEB#R0197

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Equal amounts of DNA isolated from NR-KO, iEV, and WT lines were used for E. coli transformations. For enzyme treatment of the DNA extracted from NR-KO lines, 2 μg of DNA was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 μl reaction for 1 hour at 37° C. Reactions were heat inactivated at 75° C for 30 min. An equal amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples were used to transform E. coli (DH5α high-efficiency efficiency, NEB).

Protocol tips
Equal amounts of DNA isolated from NR-KO, iEV, and WT lines were used for E. coli transformations. For enzyme treatment of the DNA extracted from NR-KO lines, 2 μg of DNA was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 μl reaction for 1 hour at 37° C. Reactions were heat inactivated at 75° C for 30 min. An equal amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples were used to transform E. coli (DH5α high-efficiency efficiency, NEB).

Protocol tips

Equal amounts of DNA isolated from NR-KO, iEV, and WT lines were used for E. coli transformations. For enzyme treatment of the DNA extracted from NR-KO lines, 2 μg of DNA was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 μl reaction for 1 hour at 37° C. Reactions were heat inactivated at 75° C for 30 min. An equal amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples were used to transform E. coli (DH5α high-efficiency efficiency, NEB).

Publication protocol 1 Relevant paper

ClaI R6551

Promega

Upstream tips	Protocol tips	Downstream tips
	Approximately 1 μg of genomic DNA from mycobacterial strains was digested overnight at 37°C with 5 U of Cla I (Promega, Madison, USA), and 10 U of Sal I. Restriction fragments were separated overnight by electrophoresis on 0.8% agarose gel and then transferred to a nylon membrane by vacuum blotting using a Milliblot-V transfer system (Millipore, Bedford, MA, USA).

Protocol tips
Approximately 1 μg of genomic DNA from mycobacterial strains was digested overnight at 37°C with 5 U of Cla I (Promega, Madison, USA), and 10 U of Sal I. Restriction fragments were separated overnight by electrophoresis on 0.8% agarose gel and then transferred to a nylon membrane by vacuum blotting using a Milliblot-V transfer system (Millipore, Bedford, MA, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

Bsu15I (ClaI) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	These plasmids were digested with ClaI and PauI (Thermo Fisher Scientific) and the Pbp1 polyglutamine sequence was gel purified and ligated into the full length Pbp1+ promoter plasmid. All plasmids had sequence fidelity confirmed by Sanger sequencing.

Protocol tips
These plasmids were digested with ClaI and PauI (Thermo Fisher Scientific) and the Pbp1 polyglutamine sequence was gel purified and ligated into the full length Pbp1+ promoter plasmid. All plasmids had sequence fidelity confirmed by Sanger sequencing.

Manufacturer protocol Publication protocol 1 Relevant paper

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