Restriction Enzymes AgeI / BshTI

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

AgeI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The backbone was digested with AgeI-HF (NEB, R3552S). Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. For cloning of single gRNAs into the lentiviral backbone, gRNA sequences were ordered as strings (see Supplementary Table 3). Strings were amplified using the libgen_fwd and libgen_rev primers. The PCR mix contained 0.1 ng DNA template, 25 µl 2x Phusion High-Fidelity PCR Master Mix with HF Buffer, 0.5 µl of each primer (100 µM), in a total reaction volume of 50 µl. The backbone was digested with AgeI-HF. Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. Transformation, PCR and plasmid preparations were done according to routine laboratory practice. gRNA sequences are listed in Supplementary Table 3.

Protocol tips
The backbone was digested with AgeI-HF (NEB, R3552S). Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. For cloning of single gRNAs into the lentiviral backbone, gRNA sequences were ordered as strings (see Supplementary Table 3). Strings were amplified using the libgen_fwd and libgen_rev primers. The PCR mix contained 0.1 ng DNA template, 25 µl 2x Phusion High-Fidelity PCR Master Mix with HF Buffer, 0.5 µl of each primer (100 µM), in a total reaction volume of 50 µl. The backbone was digested with AgeI-HF. Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. Transformation, PCR and plasmid preparations were done according to routine laboratory practice. gRNA sequences are listed in Supplementary Table 3.

Protocol tips

The backbone was digested with AgeI-HF (NEB, R3552S). Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. For cloning of single gRNAs into the lentiviral backbone, gRNA sequences were ordered as strings (see Supplementary Table 3). Strings were amplified using the libgen_fwd and libgen_rev primers. The PCR mix contained 0.1 ng DNA template, 25 µl 2x Phusion High-Fidelity PCR Master Mix with HF Buffer, 0.5 µl of each primer (100 µM), in a total reaction volume of 50 µl. The backbone was digested with AgeI-HF. Insert and backbone were gel-purified and mixed in a molar ratio of 3:1, and 2.5 µl of the mix were incubated in 2.5 µl 2x Gibson Mastermix for 30 min at 50 °C. Transformation, PCR and plasmid preparations were done according to routine laboratory practice. gRNA sequences are listed in Supplementary Table 3.

Publication protocol 1 Relevant paper

FastDigest BshTI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The vector contains a 1.2 kb stuffer sequence that was double–digested by FastDigest BshTI and EcoRI (FD1464 and FD0274; Thermo Fisher, Waltham, MA, USA) for 1 h. The 8 kb BshTI/EcoRI band was extracted from 1% agarose gel using Wizard SV Gel and PCR Clean-Up System (A9280; Promega, Madison, WI, USA).

Protocol tips
The vector contains a 1.2 kb stuffer sequence that was double–digested by FastDigest BshTI and EcoRI (FD1464 and FD0274; Thermo Fisher, Waltham, MA, USA) for 1 h. The 8 kb BshTI/EcoRI band was extracted from 1% agarose gel using Wizard SV Gel and PCR Clean-Up System (A9280; Promega, Madison, WI, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

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