OPTIZYME™ SacI, Fisher BioReagents™

Description

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

Supplied with: 10X OPTIZYME Ecl136II, SacI Buffer
Conditions for 100% Activity:

• 1X OPTIZYME Buffer Ecl136II, SacI: 10mM Bis-Tris Propane-HCl (pH 6.5 at 37°C), 10mM MgCl₂ and 0.1mg/ml BSA
• Incubate at 37°C

Enzyme Activity in OPTIZYME buffers:

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

Storage Buffer:

• 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1 mM 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 a 50-fold over-digestion with SacI.

Digestion of Agarose-embedded DNA:

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

Notes:

• SacI is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC/. AluI methyltransferase (AGmCT) can be used to block SacI.
• Supercoiled plasmids may require up to 5-fold more SacI for complete digestion than linear DNA.
• SacI is inhibited by common clinical anticoagulants found in some preparation of anticoagulated peripheral blood and bone marrow. Levels of EDTA and ACD (citric acid-sodium citrate-dextrose) in standard sample preparation have been shown to inhibit SacI. Three times the normal concentration for heparin is required to inhibit SacI.

Compatible Ends: Alw21I, Eco24I, SduI
Methylation Effects:
Dam: Never overlaps - no effect
Dcm: Never overlaps - no effect
CpG: May overlap - no effect

• 5'...m5C GAGCTm5C G...3'

3'... Gm5CTCGA Gm5C...5'

(No effect on cleavage)

EcoKI: Never overlaps - no effect

EcoBI: May overlap - no effect

• 5'...TGm6AGCTC(N)₄ TGCT...3'

3'...AC TCGAG(N)₄ m6ACGA...5'

(No effect on cleavage)