Storage

This product can be stored at -20℃ for up to 2 years.

Technical Support

R&D Department, Hangzhou Simgen Biotechnology Co., Ltd. E-mail: technical@simgen.cn, Tel: 400-0099-857.

Introduction

MinuteCut^TM enzymes are a series of engineered restriction enzymes that are capable of fast DNA digestion. All MinuteCut^TM enzymes show superior activity in the universal 10×MinuteCut^TM Buffer and 10×MinuteCut^TM Red Buffer and are able to digest DNA in 5~15 min. This enables any combination of restriction enzymes to work simultaneously in one reaction tube and eliminates the need for sequential digestions. MinuteCut^TM enzymes have passed multiple strict quality controls, and can be used to digest plasmid, genomic and viral DNA as well as PCR products. 10×MinuteCut^TM Red Buffer includes a density reagent along with red and yellow tracking dyes that allow for direct loading of the reaction mixtures on a gel. The red dye of the 10×MinuteCut^TM Red Buffer migrates with 2.5 kb double-strand DNA fragments in a 1% agarose gel, and the yellow dye migrates with 10 bp double-strand DNA fragments in a 1% agarose gel.

Quality Control

Functional Test

A 20 μl reaction in 10×MinuteCut^TM Buffer containing 1 μg of λDNA and 1 μl of MinuteCut^TM Sph Ⅰ incubated for 15 min at 37℃ results in complete digestion as determined by agarose gel electrophoresis.

Prolonged Incubation/Star Activity Assay

A 20 μl reaction in 10×MinuteCut^TM Buffer containing 1 μg of λDNA and 1 μl of MinuteCut^TM Sph Ⅰ incubated for 3 hours at 37℃ results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Longer incubation may result in star activity.

Ligation and Recutting

After 10-fold over-digestion with MinuteCut^TM Sph Ⅰ at 37℃, >95% of the DNA fragments can be ligated with T4 DNA Ligase at 22℃. Of these ligated fragments, >95% can be recut with MinuteCut^TM Sph Ⅰ as determined by agarose gel electrophoresis.

Non-specific Endonuclease Activity

A 20 μl reaction in 10×MinuteCut^TM Buffer containing 1 μg of supercoiled plasmid without the restriction site and 1 μl of MinuteCut^TM Sph Ⅰ incubated for 4 hours at 37℃ results in <10% conversion to the nicked or linearized form as determined by agarose gel electrophoresis.

Blue/White Screening Assay

An appropriate vector containing lacZα gene is digested by 1 μl MinuteCut^TM Sph Ⅰ. The digested product is ligated and transformed into E.coli competent cells. On Luria-Bertani culture plate with X-Gal, IPTG and appropriate antibiotic, the successfully ligated β-galactosidase gene can be expressed and gives rise to a blue colony, while an interrupted gene (i.e. degraded DNA end) gives rise to a white colony. MinuteCut^TM restriction enzymes must produce fewer than 1% white colonies.