OPTIZYME™ AloI, Fisher BioReagents™

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

Specifications

• Concentration: 1 to 3 U/μL
• Components: 25 to 50% Water, >50% Glycerin
• Incubator Temperature: 30°C
• pH: 7.4
• For Use With (Application): >95% of DNA fragments can be ligated and re-cut after 50-fold over-digestion with AloI
• Storage Buffer: 10mM Tris HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/mL BSA, 50% Glycerol
• Content And Storage: Keep container tightly closed
• Quantity: 100 U
• Cut Site: (7/12)GAAC(N)6TCC(12/7)
• Product Type: AloI
• Form: Liquid

Safety and Handling

SDS