Restriction Enzymes SpeI / BcuI

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 2 matching solutions for this experiment

SpeI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	‘Digestion Premix’ containing 1 μl of 10× RE solution, 3 μl of 2 M trehalose, 5.5 μl of DW, and 0.5 μl of restriction endonuclease, SpeI-HF, was added to the beads, and suspended by pipetting up and down. The suspension was incubated at 37 °C for 15 min, followed by mixing by pipetting up and down and further incubation at 37 °C for 15 min. Then, the beads were collected by magnet to remove the supernatant. The beads were rinsed twice, as described in the ‘Beads-rinsing’ section above.

Protocol tips
‘Digestion Premix’ containing 1 μl of 10× RE solution, 3 μl of 2 M trehalose, 5.5 μl of DW, and 0.5 μl of restriction endonuclease, SpeI-HF, was added to the beads, and suspended by pipetting up and down. The suspension was incubated at 37 °C for 15 min, followed by mixing by pipetting up and down and further incubation at 37 °C for 15 min. Then, the beads were collected by magnet to remove the supernatant. The beads were rinsed twice, as described in the ‘Beads-rinsing’ section above.

Protocol tips

‘Digestion Premix’ containing 1 μl of 10× RE solution, 3 μl of 2 M trehalose, 5.5 μl of DW, and 0.5 μl of restriction endonuclease, SpeI-HF, was added to the beads, and suspended by pipetting up and down. The suspension was incubated at 37 °C for 15 min, followed by mixing by pipetting up and down and further incubation at 37 °C for 15 min. Then, the beads were collected by magnet to remove the supernatant. The beads were rinsed twice, as described in the ‘Beads-rinsing’ section above.

Publication protocol 1 Relevant paper

SpeI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Their stock concentrations were 15 U/µl for EcoRV (Cat # : D1042A), 15 U/µl for PstI (Cat # : D1073A), 10 U/µl for XhoI (Cat # : D1094A), 15 U/µl for XbaI (Cat # : D1093A), 10 U/µl for SpeI (Cat # : D1086A), and 10 U/µl for SphI (Cat # : D1180A), respectively. EcoRV creates a blunt end, XbaI, SpeI and XhoI generate a 5′-overhang, and SphI and PstI create a 3′-overhang.

Protocol tips
Their stock concentrations were 15 U/µl for EcoRV (Cat # : D1042A), 15 U/µl for PstI (Cat # : D1073A), 10 U/µl for XhoI (Cat # : D1094A), 15 U/µl for XbaI (Cat # : D1093A), 10 U/µl for SpeI (Cat # : D1086A), and 10 U/µl for SphI (Cat # : D1180A), respectively. EcoRV creates a blunt end, XbaI, SpeI and XhoI generate a 5′-overhang, and SphI and PstI create a 3′-overhang.

Manufacturer protocol Publication protocol 1 Relevant paper

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