Restriction Enzymes SacI

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 5 matching solutions for this experiment

Protocol tips
The 5′ PpAOS2 genomic fragment was digested with KpnI (Takara Bio Inc., Shiga, Japan) and HindIII (Takara Bio Inc., Shiga, Japan) and inserted into pTN186, which contained a hygromycin-resistance cassette and had been digested with KpnI (Takara Bio Inc., Shiga, Japan) and HindIII (Takara Bio Inc., Shiga, Japan) to obtain pTN186-PpAOS2KO5′. The 3′ PpAOS2 genomic fragment was inserted into pTN186-PpAOS2KO5′, which had been digested with SphI (Takara Bio Inc., Shiga, Japan) and SacI (Takara Bio Inc., Shiga, Japan), to yield pTN186-PpAOS2KO.
SacI-HF®

New England BioLabs

Protocol tips
A 20 μl reaction contains:

10 μg of DNA from plasmid pFA6a-his3MX4 (this will result in ~5 μg of marker swap cassette DNA) (Lorenz, 2015a and 2015b).

2 μl CutSmart buffer

1 μl PvuII-HF restriction enzyme

1 μl SacI-HF restriction enzyme

Make up to 20 μl by adding the appropriate amount of sterile MilliQ water (see Note 1).

Incubate reaction at 37 °C for 1 h.
FastDigest SacI

Thermo Fisher Scientific

Protocol tips
The amplified PCR product was digested with SacI at the 5′ and with NheI at the 3′ end followed by ligated into SacI+NheI (FastDigest®, Fermentas, USA) digested pSY-B-MCS plasmid to yield a
construct pSY-B-GFP possessing GFP gene between the MP and CP region (Fig. 2A).
SacI (10 U/µL)

Thermo Fisher Scientific

Protocol tips
Next, the generated PCR fragment was subjected to SacI digestion (Thermo Fisher Scientific) and then blunt-sticky end ligation with pNZ8150 cut previously with SacI/ScaI enzymes.
SacI NEB#R0156

New England BioLabs

Protocol tips
PCR products were purified using GeneJet columns (Thermo-Fisher), digested with restriction enzymes BglII or SacI (NEB) and analyzed by agarose gel electrophoresis.
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