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Restriction Endonucleases >
Restriction Endonucleases >
SacI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the SacI gene from Streptomyces achromogenes (ATCC 12767).
Reagents Supplied:
NEBuffer 1
BSA
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | NEBuffer 2: | | 50% | | NEBuffer 3: | | 10% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 1
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X NEBuffer 1:
10 mM Bis-Tris-Propane-HCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.0 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (HindIII digest) in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
20,000 units/ml and 100,000 units/ml
Unit Assay Substrate:
λ DNA (HindIII digest)
Storage Conditions:
10 mM Tris-HCl
100 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- SacI is inhibited by salt concentrations > 10 mM. Mini-prep DNA containing residual salt is resistant to cleavage. A 70% alcohol wash or dialysis can be used to remove the salt.
- SacI is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC.
FAQs
- Why isn't SacI cutting?
- How can the efficiency of SacI be increased?
- Is SacI affected by methylation?
- What is the molecular weight of SacI?
- What is the activity of SacI at 25°C?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 20-fold overdigestion with SacI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with SacI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 100 units of SacI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of SacI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 60 units of SacI with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 5% conversion to RFII as determined by agarose gel electrophoresis.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
Reagents Sold Separately
NEBuffer 1
BSA
Companion Products
SacI-HF™
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,532,153
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