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 MgCl 2 , 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) 4 Gm6A TCC...3' 3'...CTTG(N) 4 C Tm6AGG...5' (Cleavage effect not determined) Dcm: May overlap - no effect 5'...GAAC(N) 6 TCm5CW GG...3' 3'...CTTG(N) 6 AG GWm5CC...5' (No effect on cleavage) CpG: May overlap - cleavage impaired 5'...m5C GAAC(N) 6 TCC...3' 3'..
• Gm5CTTG(N) 6 AGG...5' (Cleavage is blocked) 5'...GAAC(N) 6 TCm5C G...3' 3'...CTTG(N) 6 AG Gm5C...5' (No effect on cleavage) 5'...GAAm5C G(N) 5 TCC...3' 3'...CTT Gm5C(N) 5 AGG...5' (No effect on cleavage) EcoKI: Never overlaps - no effect EcoBI: Never overlaps - no effect