OPTIZYME™ NheI, Fisher BioReagents™

Description

5'...G^C T A G C...3'
3'...C G A T C^G...5'

Supplied with: 10X OPTIZYME Buffer 4
Conditions for 100% Activity:

• 1X OPTIZYME Buffer 4: 33mM Tris-acetate (pH 7.9 at 37°C), 10mM Mg-acetate, 66mM K-acetate and 0.1mg/ml BSA
• Incubate at 37°C

Enzyme Activity in OPTIZYME buffers:

• Buffer 1: 100%
• Buffer 2: 20 - 50%
• Buffer 3: 0 - 20%
• Buffer 4: 100%
• Buffer 5: 0 - 20%

Storage Buffer:

• 10mM Tris-HCl (pH 8.0 at 25°C), 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% (v/v) glycerol

Ligation and Re-cleavage:

More than 95% of the DNA fragments can be ligated and re-cut after 50-fold over-digestion with NheI.

Digestion of Agarose-embedded DNA:

A minimum of 5 units of NheI is required for the complete digestion of 1μg of agarose-embedded lambda DNA in 16 hours.

Notes:

• Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
• NheI is inhibited by salt concentrations above 100mM.
• Supercoiled plasmids may require up to 10-fold more NheI for complete digestion than linear DNAs (e.g. 10 units are required to cleave 1μg of pBR322 DNA).

Compatible Ends: XmaJI, BcuI, Eco130I, XbaI
Methylation Effects:
Dam: Never overlaps - no effect
Dcm: Never overlaps - no effect
CpG: May overlap - cleavage impaired

• 5'...GCTAGm5C G...3'

3'...CGATC Gm5C...5'

(Cleavage is impaired)

• 5'...m5C GCTAGm5C G...3'

3'... Gm5CGATC Gm5C...5'

(Cleavage is blocked)

EcoKI: Never overlaps - no effect

EcoBI: Never overlaps - no effect