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- Thermo scientific -fermentas
- ER2211
- 2025年10月06日
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|
| Catalog# | Size, concentration | Supplied with: | Certificate of Analysis | MSDS |
| 10X Buffer SgeI | ||||
| ER2211 | 250 u (3 u/µl) | 1.00 ml | ER2211 |
Reaction conditions
| Recommended buffer for 100% activity | Optimal temperature | Enzyme activity in Fermentas buffers, % | Tango™ buffer for double digestion | |||||
|---|---|---|---|---|---|---|---|---|
| B (blue) 1X |
G (green) 1X |
O (orange) 1X |
R (red) 1X |
Tango™ (yellow) 1X / 2X |
||||
| SgeI | 37°C | 0-20 | 0-20 | 0-20 | NR | NR | NR | NR |
pBR322 DNA (dcm+ )
1.4%琼脂糖
6 切割位点
100%酶活性的反应条件
1X 缓冲液SgeI：
10 mM Tris-HCl (pH 8.0, 37°C), 5 mM MgCl2 , 100 mM KCl, 0.02% Triton X-100, 0.1 mg/ml BSA。
37°C酶切。
10 mM Tris-HCl (pH 8.0, 37°C), 5 mM MgCl2 , 100 mM KCl, 0.02% Triton X-100, 0.1 mg/ml BSA。
37°C酶切。
保存缓冲液
SgeI保存在：
10 mM Tris-HCl (pH 7.4, 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA 和50% (v/v)甘油。
10 mM Tris-HCl (pH 7.4, 25°C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA 和50% (v/v)甘油。
连接和重新酶切
2倍SgeI过量酶切后，超过80%的酶切产物可以连接和重新酶切。
琼脂糖包埋DNA酶切
至少需要3 unit限制酶才能在16小时内完全切割1 µg琼脂糖包埋pBR322 dcm+ DNA。
备注
- DNA methylated by Dcm or CpG methyltransferases will be a substrate for SgeI.
- Greater than 3-fold overdigestion with SgeI may result in incomplete cleavage.
- At least two copies of SgeI recognition sequence are required for an efficient cleavage.
- Amount of the enzyme required for complete digestion of methylated DNA depends on the number of SgeI recognition sites. DNA cleavage products generated by target site cleavage facilitate the nonspecific cleavage by SgeI. Therefore, optimization of the enzyme amount is recommended for DNA cleavage.
- pBR322 DNA isolated from E.coli dcm+ strain (#SD0041) can be used as a DNA cleavage efficiency control. SgeI cleaves all six dcm methylated targets on pBR322 DNA.
商业化同裂酶
采用REsearch™软件寻找同功酶。
双酶切
采用DoubleDigest™软件确定双酶切的最佳条件。
SgeI is not recommended for double digestions.
甲基化影响
Dam: 无重叠 – 不影响。
Dcm: always cleaves DNA methylated by Dcm methyltransferase.
CpG: cleaves targets overlapping with CpG methylated sequences.
EcoKI: 无重叠 – 不影响。
EcoBI: 无重叠 – 不影响。
Dcm: always cleaves DNA methylated by Dcm methyltransferase.
CpG: cleaves targets overlapping with CpG methylated sequences.
EcoKI: 无重叠 – 不影响。
EcoBI: 无重叠 – 不影响。
| Methylation type | Sequence | Cleavage effect |
|---|---|---|
| Dcm (CCWGG) | Cm5CWGG |
|
| CpG | 5'...m5C G NG...3' |
|
| CpG | 5'...m5C G m5C G ...3' |
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