The high-quality BeyoFast™ restriction enzymes are genetically engineered for fast DNA digestion in 5-15 minutes with one buffer only. They are suitable for quick digestion of plasmid DNA, PCR products, genomic DNA, etc.

The BeyoFast™ restriction enzymes have the following advantages: (1) Digestion can be completed in 5-15 minutes. (2) All enzymes share one CutEZ™ Buffer, which simplifies the multiple enzyme digestion significantly. (3) To address the issue of varying enzyme activity in CutEZ™ Buffer, the concentration of different enzymes has been adjusted, allowing for a uniform addition of 1μl enzyme per 20μl reaction for digestion. (4) Many modifying enzymes, such as Alkaline Phosphatase, Antarctic Phosphatase, T4 DNA Ligase, T4 Polynucleotide Kinase, and T4 PNK (3′ phosphatase minus), have full activities in CutEZ™ Buffer, enabling simultaneous digestion-ligation or digestion-modification-ligation in one tube. (5) These enzymes have good activity redundancy, making it easy to handle excessive substrates or substrates that are difficult to digest.

Enzyme activity assay: At the optimal reaction temperature, 1μl of BeyoFast™ XhoI can completely digest 1μg plasmid with one single XhoI cleavage site in a 20μl reaction within 15 minutes.

Figure 1. Enzyme digestion of 1μg pCMV-Blank (4294bp) by the BeyoFast™ XhoI (D5961) in 20μl reactions with 1X CutEZ™ Buffer or 1X Easy-Load™ CutEZ™ Buffer. The reactions were incubated at 37°C for 15 minutes, then terminated by heating at 80°C for 20 minutes. The reaction products were examined by agarose gel electrophoresis anyalysis. Nucleic acids were stained using the NA-Red (EB upgraded product, 2000X). The DNA Ladder (0.2-12kb, 12 bands) was used to determine the size of DNA fragments. This figure is for reference only, which may vary depending on different experimental conditions.

Extended digestion assay: After digesting 1μg of λDNA with 1μl of BeyoFast™ XhoI for 3 hours at the optimal reaction temperature, no nuclease activity or non-specific cleavage caused by star activity was observed. However, extended digestion may result in star activity of restriction enzymes.After digesting 1μg of λDNA with 1μl of BeyoFast™ XhoI for 3 hours at the optimal reaction temperature, no nuclease activity or non-specific cleavage caused by star activity was observed. However, extended digestion may result in star activity of restriction enzymes.

Digestion-ligation-redigestion assay: At the optimal reaction temperature, the digested substrate by BeyoFast™ XhoI can be religated with the T4 DNA Ligase at 22℃. After recovering the ligation products, they can be redigested using the same restriction enzyme.

Non-specific endonuclease activity detected: At the optimal reaction temperature, incubating 1μl of BeyoFast™ XhoI with 1μg of supercoiled plasmid DNA for 4h did not change the plasmid conformation, as observed by agarose gel electrophoresis

Blue-white screening: Digest 1μl of vector containing a single lacZα gene with the BeyoFast™ XhoI, followed by re-ligation and transformation into competent Escherichia coli cells. The transformants are selected on LB agar plates containing appropriate antibiotics, IPTG, and X-gal. Clones with correctly ligated products will grow as blue colonies, while those with incorrect products are white. For the BeyoFast™ series of restriction enzymes, the percentage of white colonies should be less than 1%.

The basic information of this product is as follows:

Recognition Sequence	Isoschizomer	Working Temperature	Inactivation	Methylation Interference
5′-C^TCGAG-3′ 3′-GAGCT^C-5′	PaeR7I, TliI, BssHI, Sfr274I, SlaI, StrI	37℃	80℃ 20min	Sometimes yes

BeyoFast™ ApaLI activity in different reaction buffers (buffer compatibility) is as follows:

Beyotime CutEZ™ Buffer	Beyotime Easy-Load™ CutEZ™ Buffer	Thermo FastDigest Buffer	NEB CutSmart® Buffer	Takara QuickCut™ Buﬀer
100%	100%	100%	100%	100%

Note: The assay results are obtained in standard reactions of BeyoFastTM restriction enzymes.

Effects of methylations on BeyoFast™ ApaLI activity are as follows:

Dam	Dcm	CpG	EcoKI	EcoBI
No effect	No effect	Overlap sequence can block the cleavage	No effect	No effect

Packing List：

Item	Component	Quantity
D5961-500μl	BeyoFast™ XhoI	500μl
D6018-1ml	10X CutEZ™ Buffer	1ml
D6020-1ml	Easy-Load™ 10X CutEZ™ Buffer	1ml
Manual	–	1 copy

Storage Conditions：

Store at -20℃, stable for two years.

Precautions：

The restriction enzyme should be kept on ice during use, and stored at -20℃ immediately after use.

For ultrapure water without nuclease, we recommend the BeyoPure™ Ultrapure Water (DNase/RNase-free, Sterile).

If the expected enzyme digestion site cannot be cut, please check for potential methylation interference.

Isoschizomers may have different sensitivities to different methylations. When encountering potential methylation interference issues, a different restriction enzyme can be attempted.

This product is for R&D only. Not for drug, household, or other uses.

For your safety and health, please wear a lab coat and disposable gloves during the operation.

Ligation & Modification

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