Restriction Enzymes SmaI

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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Found 3 matching solutions for this experiment

SmaI NEB#R0141

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Protocol tips
All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Protocol tips

All enzymes used (BamHI, EcoRI, NcoI, SmaI, XbaI, and XhoI) were from New England Biolabs and had a concentration of 20 000 U/mL. The high-fidelity versions of the enzymes BamHI, EcoRI, and NcoI were employed in the experiments. Single-enzyme digestions were conducted by inserting 1 μL of enzyme for every 10 μL of sample and gently mixing via repeated pipetting. Multienzyme digestions were conducted using the same DNA-to-enzyme ratio for each individual enzyme, with 1 μL of each enzyme per 10 μL volume of sample, i.e., three enzyme digest will contain 3 μL of enzyme solution per 10 μL of total solution volume. The solutions then sat out in a room-temperature environment for a minimum of 1 h prior to analysis.

Publication protocol 1 Relevant paper

SmaI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	After treatment with proteinase K (2 mg/ml) overnight at 52°C, the sample plugs were incubated three times (for 30 min each time) with 2 ml of washing buffer B (50 mM Tris-HCl, pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride at 50°C and then rinsed three times (for 30 min each time) with 2 ml of washing buffer B. One-eighth of each plug was sliced off and digested with 10 U of SmaI (Takara Shuzo)

Protocol tips
After treatment with proteinase K (2 mg/ml) overnight at 52°C, the sample plugs were incubated three times (for 30 min each time) with 2 ml of washing buffer B (50 mM Tris-HCl, pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride at 50°C and then rinsed three times (for 30 min each time) with 2 ml of washing buffer B. One-eighth of each plug was sliced off and digested with 10 U of SmaI (Takara Shuzo)

Protocol tips

After treatment with proteinase K (2 mg/ml) overnight at 52°C, the sample plugs were incubated three times (for 30 min each time) with 2 ml of washing buffer B (50 mM Tris-HCl, pH 7.5) containing 2 mM phenylmethylsulfonyl fluoride at 50°C and then rinsed three times (for 30 min each time) with 2 ml of washing buffer B. One-eighth of each plug was sliced off and digested with 10 U of SmaI (Takara Shuzo)

Manufacturer protocol Publication protocol 1 Relevant paper

SmaI R6121

Promega

Upstream tips	Protocol tips	Downstream tips
	HindIII, EcoRI (Boehringer Mannheim), SmaI (Promega). All enzymes at 10 U μl−1. In the lower lanes (Airmail), DNA was digested with restriction enzymes that had been sent by conventional airmail in a plain envelope from Edinburgh, UK, to Boston, MA, and returned via the same route.

Protocol tips
HindIII, EcoRI (Boehringer Mannheim), SmaI (Promega). All enzymes at 10 U μl−1. In the lower lanes (Airmail), DNA was digested with restriction enzymes that had been sent by conventional airmail in a plain envelope from Edinburgh, UK, to Boston, MA, and returned via the same route.

Manufacturer protocol Publication protocol 1 Relevant paper

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