Restriction Enzymes AflII / BspTI

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 3 matching solutions for this experiment

AflII NEB#R0520

New England BioLabs

Protocol tips
For AflII and AvaII digest of cloned DNA building blocks, the RCA reaction was diluted 4-fold with water.

Digest formulations and incubation temperatures for BsrDI, AflII and AvaII (NEB) are given in Supplementary Table S2. For any digest, a mastermix of buffer, enzyme and BSA (if applicable) was created, then added to the diluted RCA reactions. After addition of enzyme mastermix to diluted RCA products, digest reaction was allowed to proceed for 4 h to ensure complete digestion, followed by heat inactivation for 20 min and finally 10-fold dilution with water. The diluted sample was analysed by capillary electrophoresis on the Fragment Analyzer instrument for verification of digest pattern.
BspTI (AflII) (10 U/µL)

Thermo Fisher Scientific

Protocol tips
To generate a pCMV-MCS-T2A-hHO1 plasmid, a pcDNA3.1-iCasper-T2A-HO1 vector (Addgene #64278) (To et al., 2015) was treated with BspTI and SgsI restriction endonucleases (Thermo Scientific) to cut out iCasper gene and then MCS (multiple cloning site) was inserted by ligation of MCS_HO1_sense and MCS_HO1_asense oligonucleotides there.
Protocol tips
The amplified fragments were digested with restriction endonucleases (ApaI, AflII, BglI, or HpaII; Takara Biomedical) and separated on 8% polyacrylamide gels, which were stained with SYBR Green I nucleic acid stain (Takara Biomedical).
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