Spe I

半岛bd体育手机客户端 说明

一般描述

SpeI recognizes the sequence *A↓*CTAGT and generates fragments with 5′-cohesive termini.

特异性

Recognition sites:*A*CTAGT
*A*CTAGT
Restriction site:*A↓*CTAGT
*A↓*CTAGT
Heat inactivation:Spe I can be heat inactivated by incubation at 65 ℃ for 15 minutes (up to 100 U/μg DNA).

质量

Absence of nonspecific endonuclease activities
1μg Ad2 DNA is incubated for 16 hours in 50μl SuRE/Cut Buffer H with an excess ofSpeI. The number of enzyme units which do not change the enzyme-specific pattern is stated in the certificate of analysis.

Absence of exonuclease activity
Approximately 5μg [3H] labeled calf thymus DNA are incubated with 3μlSpeI for 4 hours at +37℃ in a total volume of 100μl 50mM Tris-HCl, 10mM MgCl₂, 1mM Dithioerythritol, pH approximately 7.5. Under these conditions, no release of radioactivity is detectable, as stated in the certificate of analysis.

DNA图谱分析

Number of cleavage sites on different DNAs

• λ: 0
• φX174: 0
• Ad2: 3
• M13mp7: 0
• pBR322: 0
• pBR328: 0
• pUC18: 0
• SV40: 0

单位定义

One unit is the enzyme activity that completely cleaves 1 μg Ad2 DNA in one hour at+37 ℃in a total volume of 25 μl SuRE/Cut BufferH.

分析说明

Compatible ends
SpeI ends are compatible with ends generated byBlnI,NheI, andXbaI.

Isoschizomers
The enzyme is an isoschizomer ofBcuI andAhlI.

Methylation sensitivity
As indicated by (*) on the recognition sequence above,SpeI is inhibited by the presence of N6-methyladenine and 5′-methylcytosine (^mA↓^mCTAGT).

Incubation temperature
+37℃

PFGE tested
SpeI has been tested in Pulsed-Field Gel Electrophoresis (on bacterial chromosomes). For cleavage of genomic DNA (E. coliC 600) embedded in agarose for PFGE analysis, we recommend using 10U of enzyme/μg DNA and 4 hour incubation time.

Ligation and recutting assay
SpeI fragments obtained by complete digestion of 1μg Ad2 DNA are ligated with 1U T4 DNA Ligase in a volume of 10μl by incubation for 16 hours at +4℃ in 66mM Tris-HCl, 5mM MgCl₂, 5mM Dithiothreitol, 1mM ATP, pH 7.5 (at +20℃), resulting in >90% recovery of Ad2 DNA.
Subsequent re-cutting withSpeI yields >95% of the typical pattern of Ad2 ×SpeI fragments.

SuRE/Cut Buffer System
The buffer inboldis recommended for optimal activity

• A: 75-100%
• B: 75-100%
• H: 100%
• L: 75-100%
• M: 100%

Activity in PCR buffer: Not tested

其他说明

仅用于生命科学研究。不可用于诊断。

半岛bd体育手机客户端 性质

质量水平	100
生物来源	bacterial (Sphaerotilusspp.)
形式	solution
包装	pkg of 1,000 U (11008951001 [10 U/μl]) pkg of 1,000 U (11207644001 [40 U/μl]) pkg of 200 U (11008943001 [10 U/μl])
manufacturer/tradename	Roche
参数	37 ℃ optimum reaction temp.
运输	dry ice
储存温度	?20℃

组份列表

组份不可单独销售

货号	组份
	Enzyme Solution
	SuRE/Cut Buffer H 10x concentrated

安全信息

储存分类代码	12 - Non Combustible Liquids
WGK	WGK 1
闪点(F)	does not flash
闪点(C)	does not flash