Restriction Enzymes AsiSI / SfaAI / SgfI

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 4 matching solutions for this experiment

AsiSI NEB#R0630

New England BioLabs

Protocol tips
Individual libraries were blunt‐ended using a PCRTerminator® end repair kit (Lucigen, http://www.lucigen.com), digested with either AscI or AsiSI (NEB, http://www.neb.com), and ligated into the pre‐cut binary vector (pSR486). pSR486 was pre‐digested with either AscI and PmeI (for sense library orientation) or with AsiSI and BstZ17I (for antisense orientation), followed by dephosphorylation using Antarctic phosphatase (NEB).
SgfI R7103

Promega

Protocol tips
The amplified ORF and the pTUNE inducible vector (OriGene Technologies, Inc.) were digested with endonuclease XhoI (New England Biolabs, Inc., Beijing, China) and SgfI (Promega Corporation, Madison, WI, USA).
The CDKN2A/p16-wild-type ORF was subsequently digested by the restriction enzymes XhoI and SgfI to construct the pTUNE-CDKN2A/p16-wild-type inducible vector. The positive clone was also sequenced for verification.
FastDigest SfaAI

Thermo Fisher Scientific

Protocol tips
Prior to the USER reaction, the parental vectors were digested with FastDigest AsiSI (Thermo Fisher Scientific) and nicked with Nb. BsmI (New England Biolabs). The USER reactions were transformed into chemically competent E. coli DH5α. Correct assembly was verified by sequencing.
SfaAI (AsiSI) (10 U/µL)

Thermo Fisher Scientific

Protocol tips
The CDKN2A/p16-wild-type ORF was subsequently digested by the restriction enzymes XhoI and SgfI to construct the pTUNE-CDKN2A/p16-wild-type inducible vector. The positive clone was also sequenced for verification.
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