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SkanIt 软件培训教程
ThermoFisher Scientific Sep. 2009
Lesson 1 Hello, SkanIt!
你将学习：
• 以比色测量为例 • 创建程序 (session) • 定义检测区域 (plate layout) • 设置数据自动导出
Average rate
Decreasing max. rate Increasing max. rate
Calculation step: Kinetic Processors
Time to maximum rate /2: calculates the max. rate, calculates max. rate /2 and then identifies when max. rate /2 takes place
• 化学发光光谱扫描 • 半带宽计算 • 数据文件：LU_Scan
带宽选择
荧光激发波长和发射波长的间隔 必须大于激发和发射带宽之和：
• 使用5 nm激发带宽和12 nm发射带 宽（固定），激发波长和发射波 长的间隔必须大于17nm(实际大于 20nm)
• 使用12nm激发带宽和12nm发射带 宽（固定），激发波长和发射波 长的间隔必须大于24nm（实际大 于27nm）
• Parameters: Readings (0...100): & First/Last
Lesson 7 时间分辨荧光检测
你将学习：
• TRF 检测原理 • TR-FRET (LanthaScreen) 检测原理 • TR-FRET检测过程的实现
• 比率测量
• 数据处理过程包括：
• 扣减本底 • 自定义方程 • 曲线拟合
Time to Maximum (Peak) /2; identifies the max. value, calculates the half of the max. value and returns the time when the half max. value was measured
数据文件：LanthaScreen.txt 读板方向：7
TRF 时间分辨荧光
TRF (Time Resolved Fluorescence)
• 使用特殊的镧系元素进行标记，荧光寿命延长至μs级 • 而普通荧光寿命仅为ns级，如Fluorescein 3.6ns • 经一定的时间延迟，待背景荧光信号淬灭后，进行标
• 数据文件导入与导出 (msd文件)
• Import/Export 命令
Lesson 3 定义样品类型
你将学习：
• 样品类型包括：
• 标准品 (Calibrator) • 质控品 (Control) • 待测样品 (Unknown) • 空白 (Blank) • 无样品 (Empty)
• 孔板布局的填充向导
Calculation step: Kinetic Processors
Average rate: calculates the average rate of all measurement points in each well
Maximum rate: calculates the max. rate in each well
Ignore readings: leave out certain klinetic time points
Integral: calculates the area under the kinetic curve
Sum: adds upp the kinetic readings from all kinetic time points
2. Prepare 70% ethanol as wash reagent. Prime the system with at least 5ml of 70% ethanol to completely replace the void volume and rinse the injector plumbing. It is preferable to allow the injector to soak in this wash solution for 30 minutes prior to rinsing with deionized water.
Maximum – Minimum (Change): calculates the difference between the max. and min. measurement values in each well
Time to Maximum (Peak): identifies the maximum value in each well and returns the time when it was measured
24 nm
12 nm
12 nm
12 nm 12 nm
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• Sum = Integral / kinetic interval
Select reading
Calculation step: Kinetic Processors
Average value: calculates the average value of all kinetic readings
3. Rinse with a volume of deionized water equivalent to at least 3 pump void volumes to thoroughly remove all traces of ethanol.
(According to Promega’s Manual)
• Undefined, Increasing and Decreasing
Time to change: calculates the time it takes for an absolute (in Abs, RFU or RLU) or relative (%) change to take place compared to a basline
• 浓度系列向导 (Concentration) • 稀释度系列向导 (Dilution)
• 直接定义与修改
• 修改样品类型 • 添加样品详细信息 (Detail) • 孔板布局的Copy, Paste和Clear
Lesson 4 标准曲线计算
你将学习：
• 曲线拟合的方法
• 曲线拟合的类型 • 数据转换 (线性和对数) • 标准曲线保存 • 标准曲线外推
记物的荧光强度测量
Settings for Europium Assay
FRET 荧光共振能量转移原理
Fluorescence Resonance Energy Transfer (FRET)
• 两个荧光标记，一个作为能量供体 (Donor)，另一个作为能量受体 (Acceptor)
• 能量供体的发射光谱和受体的激发光 谱重叠
• Undefined, Increasing or Decreasing max rate
Time to maximum rate: calculates the max rate and identifies when it occurrs
• Time to Undefined, Increasing or Decreasing max rate
• Parameters: Ignore first (n) readings & Ignore last (n) readings)
Basline subtraction: subtracts the average of a defined number of readings from the beginning or the end from all other readings
Dispenser Washing Protocol for DLR
1. Purge Stop & Glo Reagent from the injector lines by repeated priming/washing with a volume of deionized water equivalent to 3 pump void volumes.
Time to Maximum
Maximum – Minimum
Calculation step: Kinetic Processors
Select reading: selects a certain kinetic time point for further data reduction
Lesson 6 动力学检测
你将学习：
• 孵育的方法 • 动力学过程(Kinetic Loop)
• 检测波长：405 nm • 动力学间隔：150 s • 动力学点：40
• 动力学数据计算
• Time to Change：0.2 OD • 直线拟合，XY作对数变换
• 动力学数据导出
Kinetic QCL kit from LONZA，数据文件：Endotoxin.txt 读板方向：7
• 当供体与受体充分接近时，发生能量 转移
• 当使用的Donor的荧光标记是时间分 辨的，即为TR-FRET
Lesson 8 光谱扫描
你将பைடு நூலகம்习：
• 比色光谱扫描 • 光谱数据计算 • 光谱数据导出 • 数据文件：TMB_Scan