SpeedyCut PacI

Description

Restriction digestion workflows can become time-consuming when different enzymes require separate reaction buffers, sequential digestion steps, or repeated buffer exchange. These additional steps can complicate plasmid DNA preparation, PCR product processing, genomic DNA digestion, and other molecular cloning workflows.

SpeedyCut PacI is a fast PacI restriction enzyme designed to cleave DNA using the universal SpeedyOne Buffer. The SpeedyCut restriction enzyme system supports rapid DNA digestion while allowing compatible enzymes from the series to be combined in a unified reaction system.

SpeedyCut PacI recognizes the sequence 5′-TTAATTAA-3′ and is suitable for restriction digestion of plasmid DNA, PCR products, and genomic DNA. Under the stated unit-definition conditions, 1 μL of enzyme completely digests 1 μg of pPacI DNA in a 20 μL reaction at 37°C within 15 minutes.

The enzyme can also be used for direct digestion of PCR reaction mixtures after amplification. Following digestion, SpeedyCut PacI may be heat-inactivated at 80°C for 20 minutes when enzyme inactivation is required.

Specifications

Features

• Rapid PacI restriction digestion: supports complete digestion under the stated unit-definition conditions within 15 minutes.
• Universal SpeedyOne Buffer: SpeedyCut restriction enzymes use a common reaction buffer to simplify restriction enzyme workflows.
• Multiple DNA substrates: suitable for digestion of plasmid DNA, PCR products, and genomic DNA.
• Direct PCR product digestion: PCR reaction mixtures can be digested directly after amplification using the recommended protocol.
• Double- and multiple-digestion support: compatible SpeedyCut enzymes can be combined in an appropriately adjusted reaction system.
• Heat inactivation: enzyme activity can be stopped by incubation at 80°C for 20 minutes.
• Quality-controlled enzyme performance: evaluated using long-incubation, ligation, recleavage, exonuclease, nonspecific endonuclease, and blue/white cloning assays.

Applications

Plasmid DNA Digestion

SpeedyCut PacI can be used for rapid restriction digestion of plasmid DNA. The recommended incubation time for plasmid substrates is 15 minutes at 37°C in 1X SpeedyOne Buffer.

PCR Product Digestion

The enzyme is suitable for restriction digestion of purified PCR products. A reaction time of 15–30 minutes at 37°C is recommended for PCR product digestion.

Direct Digestion After PCR Amplification

SpeedyCut PacI may be added directly to a PCR reaction mixture after amplification. This workflow allows PCR product digestion without first preparing a separate purified DNA substrate, using the reaction composition described in the product manual.

Genomic DNA Digestion

SpeedyCut PacI can be used for restriction digestion of genomic DNA. Because genomic DNA may require a longer incubation period, the recommended digestion time is 30–60 minutes at 37°C.

Restriction Digestion in Molecular Cloning Workflows

PacI-cleaved DNA may be used in workflows that include DNA digestion, recovery, ligation, transformation, and subsequent analysis. Product quality control includes cleavage, ligation, recleavage, and blue/white cloning assays.

Double and Multiple Restriction Digestion

SpeedyCut PacI may be used with other compatible SpeedyCut restriction enzymes in a shared SpeedyOne Buffer reaction system. Reaction volume and enzyme volume should be adjusted according to the product instructions.

Recommended DNA Digestion Protocol

The following reaction is based on the 20 μL digestion system described in the product manual.

1. Add the reaction components in the amounts shown above.
2. Mix gently and briefly spin down the reaction tube.
3. Incubate at 37°C for 15 minutes for plasmid DNA, 15–30 minutes for PCR products, or 30–60 minutes for genomic DNA.
4. When required, incubate at 80°C for 20 minutes to heat-inactivate the enzyme and stop the reaction.

Direct Digestion of PCR Products After Amplification

1. Combine the PCR reaction mixture with nuclease-free water, SpeedyOne Buffer, and SpeedyCut PacI.
2. Mix gently and briefly spin down.
3. Incubate at 37°C for 15–30 minutes.

Double and Multiple Digestion Guidelines

• Use 1 μL of each selected enzyme and enlarge the total reaction volume as needed.
• The combined volume of all enzymes must not exceed one-tenth of the total reaction volume.
• When the selected restriction enzymes have different optimal reaction temperatures, first incubate with the enzyme that has the lower optimal temperature. Then add the enzyme requiring the higher temperature and continue incubation at the higher temperature.

Quality Control

Long-Incubation and Star Activity Testing

No nonspecific degradation caused by contaminating nucleases or star activity was detected after 1 μL of SpeedyCut PacI was incubated with 1 μg of pPacI DNA for 3 hours. The manual notes that star activity may occur with longer incubation.

Cleavage, Ligation, and Recleavage Assay

DNA substrate was digested using a tenfold enzyme amount and the digested product was recovered. More than 95% of the digested product could be religated using an appropriate amount of T4 DNA ligase at 22°C. After recovery of the ligation product, more than 95% could be cleaved again using the same restriction endonuclease.

Exonuclease Activity Assay

In a 50 μL reaction system, less than 0.1% radioactivity was released after a tenfold amount of enzyme was incubated with 1 μg of ultrasonically fragmented, radiolabeled DNA at 37°C for 4 hours.

Nonspecific Endonuclease Activity Assay

After a tenfold amount of enzyme was incubated with 1 μg of supercoiled DNA for 4 hours, the plasmid DNA remained in a supercoiled state when evaluated by agarose gel electrophoresis.

Blue/White Cloning Assay

The SpeedyCut restriction enzyme series is tested using a blue/white cloning assay involving digestion, religation, transformation into competent E. coli, and colony evaluation. The specified white-colony ratio is less than 1%.

Enzyme Properties

Number of PacI Recognition Sites in Reference DNA

Storage

Store SpeedyCut PacI at −20°C.

Documents

SpeedyCut PacI Product Manual

SpeedyCut PacI Safety Data Sheet

Frequently Asked Questions

Research Use Only. This product is designed for scientific research and in vitro use only. It is not tested or intended for diagnostic or therapeutic procedures, administration to humans or animals, or use as food, cosmetics, or household products.