Restriction Enzymes AlwI / BspPI

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

BspPI (AlwI) (2 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The final PCR product from amplification of PON1 was subjected to digestion by BspPI Restriction Endonuclease (RE) enzyme (Thermo Scientific). A 10 μL of PCR reaction mixture was mixed with 2 U/μL of BspPI RE enzyme, 2 μL of 10X Buffer Tango and 18 μL of NFW. The mixture was incubated at 55°C for 16 hours. The enzyme was then inactivated by incubation at 80°C for 20 minutes. The digested product was visualised after electrophoresis in Ethidium Bromide (EtBr)-stained 2.5% agarose gel on gel documentation system (UVI-TEC).

Protocol tips
The final PCR product from amplification of PON1 was subjected to digestion by BspPI Restriction Endonuclease (RE) enzyme (Thermo Scientific). A 10 μL of PCR reaction mixture was mixed with 2 U/μL of BspPI RE enzyme, 2 μL of 10X Buffer Tango and 18 μL of NFW. The mixture was incubated at 55°C for 16 hours. The enzyme was then inactivated by incubation at 80°C for 20 minutes. The digested product was visualised after electrophoresis in Ethidium Bromide (EtBr)-stained 2.5% agarose gel on gel documentation system (UVI-TEC).

Protocol tips

The final PCR product from amplification of PON1 was subjected to digestion by BspPI Restriction Endonuclease (RE) enzyme (Thermo Scientific). A 10 μL of PCR reaction mixture was mixed with 2 U/μL of BspPI RE enzyme, 2 μL of 10X Buffer Tango and 18 μL of NFW. The mixture was incubated at 55°C for 16 hours. The enzyme was then inactivated by incubation at 80°C for 20 minutes. The digested product was visualised after electrophoresis in Ethidium Bromide (EtBr)-stained 2.5% agarose gel on gel documentation system (UVI-TEC).

Manufacturer protocol Publication protocol 1 Relevant paper

AlwI NEB#R0513

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The PCR products were separately digested with the corresponding restriction enzyme (MboII for 3′-half and AlwI for 5′-half) and then ssDNAs were collected through avidin-biotin binding (streptavidin-coated magnetic beads were used)

Protocol tips
The PCR products were separately digested with the corresponding restriction enzyme (MboII for 3′-half and AlwI for 5′-half) and then ssDNAs were collected through avidin-biotin binding (streptavidin-coated magnetic beads were used)

Publication protocol 1 Relevant paper

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