SacI
Detailní popis
Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.
Highlights
- Superior quality – stringent quality control and industry leading manufacturing process
- Convenient color-coded Five Buffer System
- Includes universal Tango buffer for double-digestions
- BSA premixed in reaction buffers
- Wide selection of restriction endonuclease specificities
Applications
- Molecular cloning
- Restriction site mapping
- Genotyping
- Southern Blot
- Restriction fragment length polymorphism (RFLP)
- SNP
Note
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. (J. E. Coad et al., Inhibition of restriction endonucleases by common clinical anticoagulants. Anal. Biochem. 205, 368-369, 1992).
For methylation sensitivity refer to product specifications.
| Compatible Ends | Alw21I, Eco24I, SduI. |
| Conditions for 100% Activity |
|
| Digestion of Agarose-embedded DNA | Minimum 5 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded lambda DNA in 16 hours. |
| Double Digestion | Perform double digestion using DoubleDigest. |
| Hazardous | No |
| Isoschizomers | Search for commercial isoschizomers using REsearch. |
| Storage Buffer | SacI is supplied in: 10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mg/mL BSA, and 50% (v/v) glycerol. |
| Storage Condition | -20 C |
Reaction conditions
| Recommended buffer for 100% activity | Optimal temperature | Enzyme activity in Thermo Scientific buffers, % | Tango buffer for double digestion | |||||
|---|---|---|---|---|---|---|---|---|
| B (blue) 1X |
G (green) 1X |
O (orange) 1X |
R (red) 1X |
Tango (yellow) 1X / 2X |
||||
| SacI Buffer (Unique) | 37°C | 50-100 | 20-50 | 0-20 | 0-20 | 50-100 | 20-50 | 1X or 2X |
Methylation Effects
- Dam: never overlaps - no effect.
- Dcm: never overlaps - no effect.
- CpG: may overlap - no effect.
- EcoKI: never overlaps - no effect.
- EcoBI: may overlap - no effect.
| Methylation type | Sequence | Cleavage effect |
|---|---|---|
| CpG | 5'...m5C GAGCTm5C G...3'3'... Gm5CTCGA Gm5C...5' |
No effect |
EcoBI (TGA(N)8TGCT) |
5'...TGm6AGCTC(N)4 TGCT...3'3'... AC TCGAG(N)4m6ACGA...5' |
No effect |
Cleavage efficiency close to the termini of PCR fragments
| bp from the recognition site to fragment end | ||||
|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 |
| 50-100 | ||||
Number of recognition sites in DNA molecules
| Lambda | ΦX174 | M13mp18/19 | pBR322 | puc18/19 | pUC57 |
|---|---|---|---|---|---|
| 2 | 0 | 1 | 0 | 1 | 1 |
| pTZ19R/U | pTZ57R | pBluescriptIIKS(±) | pBluescriptIISK(±) | pACYC177 | pACYC184 |
| 1 | 1 | 1 | 1 | 0 | 0 |
New sites generated by ligation
Newly generated recognition sites resulting from ligation of protruding compatible DNA ends
| Recognition Sequence | Second Enzyme | Enzymes recognizing newly generated recognition sequence |
|---|---|---|
GAGCT^C |
|
|
|
|
Hodnocení produktu
Produkt zatím nikdo nehodnotil, buďte první!