PvuII

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Catalog Size Concentration
R0151L 25,000 units 10,000 units/ml
R0151M 25,000 units 50,000 units/ml
R0151S 5,000 units 10,000 units/ml
R0151T 5,000 units 50,000 units/ml

Recognition Site:

IMAGEM

Description:
PvuII has a High Fidelity version PvuII-HF™ (NEB #R3151).

High Fidelity (HF) Restriction Enzymes have been engineered for reduced star activity and have 100% activity in buffer 4 which may simplify your double digest.

Source:
A E. coli strain that carries the PvuII gene from Proteus vulgaris (ATCC 13315).

Reagents Supplied:
NEBuffer 2

Enzyme Properties
Activity in NEBuffers:
NEBuffer 1:        100%
NEBuffer 2:        100%
NEBuffer 3:        100%
NEBuffer 4:        100%

When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.

Methylation Sensitivity:
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Not sensitive
More information about: Methylation Sensitivity

Heat Inactivation:
No

Survival in a Reaction:
(+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours.
More information about: Extended Digests with Restriction Enzymes

Reaction & Storage Conditions

Reaction Conditions:
1X NEBuffer 2
Incubate at 37°C.

1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C

Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

Concentration:
10,000 units/ml and 50,000 units/ml

Unit Assay Substrate:
λ DNA

Storage Conditions:
10 mM Tris-HCl
300 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Storage Temperature:
-20°C

Diluent Compatibility:
Diluent B

Notes
General notes:

1. Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.

Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.

Ligation and Re-cutting:
After a 10-fold overdigestion with PvuII, > 95% of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM) at 16ºC. Of these ligated fragments, > 95% can be recut with PvuII.

16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA and 10 units of PvuII incubated for 16 hours at 37ºC resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.

Exonuclease Activity:
Incubation of a 50 μl reaction containing 500 units of PvuII with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity.

Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of PvuII with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 10% conversion to RFII as determined by agarose gel electrophoresis.

IMAGEM

vuII crystals (Ira Schildkraut and Joseph Bonventre, New England Biolabs)

Reagents Sold Separately
NEBuffer 2

Companion Products
PvuII-HF™


PvuII FAQ

Q1: What is Star Activity and how can it be avoided?
A1: It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed "star" activity. It has been suggested that star activity may be a general property of restriction endonucleases and that any restriction endonuclease can be made to cleave noncanonical sites under certain extreme conditions.

The manner in which an enzyme's specificity is altered depends on the enzyme and on the conditions employed to induce the star activity. The most common types of altered activity are single base substitutions, truncation of the outer bases in the recognition sequence, and single-strand nicking.

Star activity is completely controllable in the vast majority of cases and is generally not a concern when performing restriction endonuclease digests. New England Biolabs' enzymes will not exhibit star activity when used under recommended conditions in their supplied NEBuffers. Listed below are reaction conditions known to induce or inhibit star activity.

Conditions that Contribute to Star Activity
1. High glycerol concentration [> 5% v/v]
2. High units to µg of DNA ratio [Varies with each enzyme, usually >100 units/µg]
3. Low ionic strength [< 25 mM]
4. High pH [> pH 8.0]
5. Presence of organic solvents [DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane]
6. Substitution of Mg++ with other divalent cations [Mn++, Cu++, Co++, Zn++]

Inhibiting Star Activity
If you are concerned about star activity, we recommend the following guidelines.
1. Use as few units as possible to get a complete digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction.
2. Make sure the reaction is free of any organic solvents such as alcohols which might be present in the DNA preparation.
3. Raise the ionic strength of the reaction buffer to 100?150 mM (provided the enzyme is not inhibited by high salt).
4. Lower the pH of the reaction buffer to pH 7.0.
5. Use Mg++ as the divalent cation.

Q2: How can this enzyme be inactivated?
A2: To inactivate enzymes that cannot be heat killed, we recommend a phenol extraction followed by ethanol precipitation or a commercial spin column designed to purify DNA.

 
 

 

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