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OPTIZYME™ AloI, Fisher BioReagents™

Catalog No. BP8075-1

Item Discontinued This item has been discontinued by Fisher Scientific. Please to view product availability in your area.


Description

Description

5'...^ 7(N) G A A C (N)6 T C C (N)12-13^...3'
3'...^12-13(N) C T T G (N)6 A G G (N)7 ^...5'

Supplied with: 10X OPTIZYME Buffer 5
Conditions for 100% Activity:

  • 1X OPTIZYME Buffer 5: 10mM Tris-HCl (pH 8.5 at 37°C), 10mM MgCl2, 100mM KCl and 0.1mg/mL BSA
  • Incubate at 30°C
Enzyme Activity in OPTIZYME buffers:
  • Buffer 1: 0 - 20%
  • Buffer 2: 0 - 20%
  • Buffer 3: 0 - 20%
  • Buffer 4: 20 - 50%
  • Buffer 5: 100%
Storage Buffer:
  • 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/mL BSA and 50% (v/v) glycerol
  • Ligation and Re-cleavage:
  • Approximately 70% of the DNA fragments can be ligated and more than 80% of these can be re-cut after 10-fold over-digestion with AloI.
  • Notes:
    • Incubation at 37°C results in 20% activity.
    • AloI produces double-strand cuts on both sides from the interrupted recognition site. Its unique feature is a degenerate cleavage point on the 3 side of the recognition sequence (12 or 13 nucleotides away).
    • The presence of S-adenosylmethionine in the reaction mixture results in incomplete cleavage with AloI.
    • Greater than 10-fold overdigestion with AloI may result in star activity.
    • AloI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
    Methylation Effects:
    Dam: May overlap - effect not determined
    • 5'...GAAC(N)4Gm6A TCC...3'
  • 3'...CTTG(N)4C Tm6AGG...5'
  • (Cleavage effect not determined)
  • Dcm: May overlap - no effect
    • 5'...GAAC(N)6TCm5CW GG...3'
  • 3'...CTTG(N)6AG GWm5CC...5'
  • (No effect on cleavage)
  • CpG: May overlap - cleavage impaired
    • 5'...m5C GAAC(N)6TCC...3'
  • 3'... Gm5CTTG(N)6AGG...5'
  • (Cleavage is blocked)
    • 5'...GAAC(N)6TCm5C G...3'
  • 3'...CTTG(N)6AG Gm5C...5'
  • (No effect on cleavage)
    • 5'...GAAm5C G(N)5TCC...3'
  • 3'...CTT Gm5C(N)5AGG...5'
  • (No effect on cleavage)
  • EcoKI: Never overlaps - no effect
  • EcoBI: Never overlaps - no effect
  • TRUSTED_SUSTAINABILITY
    Specifications

    Specifications

    1 to 3 U/μL
    30°C
    >95% of DNA fragments can be ligated and re-cut after 50-fold over-digestion with AloI
    Keep container tightly closed
    (7/12)GAAC(N)6TCC(12/7)
    Liquid
    25 to 50% Water, >50% Glycerin
    7.4
    10mM Tris HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/mL BSA, 50% Glycerol
    100 U
    AloI
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