For general laboratory use.

Unit Definition: One unit is the amount of enzyme required to completely digest 1 μg of Lambda DNA (5 sites) in 1 hour in a total reaction volume of 50 μl. Enzyme activity was determined in the recommended reaction buffer.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Form: liquid (Supplied in 10 mM Tris-HCl pH 7.4, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50 % [v/v] glycerol)

Concentration: 10 units/μl

Source: Bacillus amyloliquefaciens H

Supplied with: 10x Universal Buffer (UB)

Recommended 50 μl assay
¹ Supercoiled or high molecular weight DNA (e.g. plant genomic DNA) may require longer incubation time or higher amount of enzyme.
² Some enzymes may require additional DNA bases flanking the restriction site for complete digestion.

Protocol:

• The enzyme should not exceed 10 % of total reaction volume.
• Add enzyme as last component. Mix components well before adding enzyme. After enzyme addition, mix gently by pipetting. Do not vortex.
• Incubate 5 to 10 min. at 37 °C.
• Stop reaction by alternatively:
  - Addition of 2.1 μl EDTA pH 8.0 [0.5 M], final 20 mM
  - Heat Inactivation (20 min. at 80 °C)
  - Spin Column DNA Purification (e.g. PCR Purification Kit, Cat.-No. PP-201S/L)
  - Gel Electrophoresis and Single Band Excision (e.g. Agarose Gel Extraction Kit, Cat.-No. PP-202S/L)
  - Phenol-Chloroform Extraction or Ethanol Precipitation.

Double Digestion - Buffer Compatibility:
B1 - 75 % Relative Activity
B2 - 75-100 % Relative Activity
B3 - 100 % Relative Activity
B4 - 50-75 % Relative Activity
B5 - 75 % Relative Activity
1x UB - 100 % Relative Activity (recommended)

Please note that the optimum digestion condition for this enzyme is 1x UB. Within the Universal Buffer (UB) system, the most majority of our enzymes display 100% Relative Activity in 1x UB and only few either in 0.5x or 2x UB. If optimum condition for second enzyme is different than the recommended for the first enzyme, we suggest carrying out first the restriction at the higher recommended concentration of UB and dilute the reaction volume to the adequate UB concentration for further proceeding with the second restriction.

Reaction Enzymes Buffer Guide:

Reaction Buffer Compatibility:
Our restriction enzymes are fully compatible to restrictases and buffer systems from other manufacturers and can be used along in double digestions. To obtain best results, consult the corresponding manuals of all involved products.

Ligation and recutting:
After 50-fold overdigestion with BamHI, >95 % of the DNA fragments can be ligated and recut with this enzyme.

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

DNA Methylation:
No Inhibition: dam, dcm, CpG

Quality Control:
All preparations are assayed for contaminating endonuclease, 3'-exonuclease, 5' exonuclease/ 5' phosphatase, as well as nonspecific single- and doublestranded DNase activities.