美国Bio-rad伯乐CHEF Mapper XA脉冲场电泳仪
CHEF MAPPER XA 是目前的脉冲场电泳系统，拥有设计的六角形电极的电泳槽，保证矢量电场的自由旋转，比常规的脉冲场电泳提供更高的分辨率，电泳速度和更精确的分离，对100bp-10Mb的DNA片段都能提供有效的分离。主要用于基因组DNA的分离分析，大分子DNA的指纹图谱分析及多态性分析等。
1．自动演算：Bio-Rad积多年在脉冲场电泳方面的经验，提供给用户一套程序用于优化实验参数。只要输入待分离DNA片段的最小、长度，结合10个主要变量的确定，帮助使用者获得的实验条件。
2．脉冲角度：可以在0-360°间自由选择脉冲角度，使用户可以在同一系统上实现大至染色体级、小至质粒DNA的有效分离。
3．时间转换梯度：有线性、非线性（凸形和凹形）两种脉冲时间梯度，非线性梯度可以提供更广泛的分离动态范围，使用户可以精确的确定分离片段的大小。
4．多状态功能：CHEF Mapper XA在一个Block中可以有15个电场矢量，每个电场矢量可以有自己的电压和转换时间，可有选择地对一定大小范围的片段进行更精细的分离，并且可以在同一次电泳中实现FIGE和CHEF两种技术。
5．二次脉冲功能：二次脉冲可加速DNA从琼脂糖凝胶中释放，从而有利于非常大的DNA片段的分离，并可提高分辨率。
6．技术应用：
CHEF（钳式均衡电场）技术，产生均衡电场；
PACE（程序自主控制电极）技术，根据片断大小的需要优化设定脉冲角度；
FIGE（电场倒置）技术，为 250KB以下小片断DNA提供快速分离；
AFIGE（非对称场倒置）技术，精细分离小片断DNA，提高分辨率；
以上技术的应用保证了科研人员在所有分子量范围内均能得到所的线性分离。
性能指标：
a. 电源输出：电压 350V，0和0.6-9V/cm，0.1V/cm增量，连续可调
b. 电流：0.5A
c. 延迟启动：72小时
d. 电极调节能力：动态调节（反馈调整）±0.5％
e. 程序储存：存储20个复杂实验程序，每个程序包含8个程序模块或99个简单程序
f. 数据记录：键盘，条形码读取或RS-232接口
g. 显示屏：2行×40字符/行，荧光显示
h. 转换范围：50毫秒到18小时
i. 转换角度：0-360°，0.5°增量
j. 电泳时间：999小时/模块
脉冲中断设置：可以通过电压，频率，角度和持续时间设定
The CHEF Mapper system lets you choose any pulse angle from 0–3,600. This allows you to achieve optimal separation of both chromosomal DNA (Figure 1) and plasmid DNA (Figure 2), with one flexible system.
A, 106° angle; B, 120° angle.
Two-state mode
48 hr run
2 V/cm (67 V), 14°C, 1x TAE
30 min switch time
0.8% chromosomal-grade agarose
Fig. 1, Increased mobility of S. pombe chromosomes using the CHEF Mapper XA system
FIGE mode
180° angle
1x TAE, 14°C
9 V/cm forward
6 V/cm reverse
18 hr run
Switch time 200–800 ms ramp
Forward switch time = reverse time
Lane 1: Bio-Rad lambda HindIII standard (6.6, 9.4, 23.1 kb)
Lane 2: Bio-Rad 8–48 kb size standard (8.3, 8.6, 10.0, 12.2, 15.0, 17.1, 19.4, 22.6, 24.8, 29.9, 33.5, 38.4, 48.5 kb)
Fig. 2, High resolution of 8–48 kb size standard on the CHEF Mapper XA system with asymmetric voltage FIGE.
Accurate sizing of fragments requires an expanded linear range of separation. Switch-time ramps increase the mobility of fragments as a function of molecular weight by gradually changing the switch times during the course of a run. Nonlinear ramps change the rate at which the switch time moves from the specified initial switch time to the specified final switch time. These nonlinear ramps (e.g., concave or convex) have been shown to provide very linear separations from 50–700 kb (Figure 3). Therefore, fragment sizes will be measured more precisely.
Fig. 3. Mobility effects of nonlinear switch time ramps on the CHEF Mapper system. Molecular size vs. migration for linear, concave, and convex ramps. The convex ramp results in the widest linear range.
The multi-state mode of the CHEF Mapper system enhances resolution in selected fragment size ranges. Each vector (angle of pulse) can be assigned its own voltage (field intensity) and its own switch time (duration of pulse). Up to eight different states may be combined into one run to optimize the separation of subsets of fragments in the sample (Figure 4). The application of secondary pulses of defined voltage, duration, angle, and frequency can enhance the separation and resolution of very large DNA molecules (Figure 5). These secondary pulses may release DNA caught in the gel matrix.
A. Two-state mode
24 hr run time, 120° included angle
60 to 120 sec switch-time ramp
6 V/cm, 0.5x TBE at 14°C
1.0% pulsed field Certified agarose
B. Multi-state mode
60 hr run time
State (vector)
1. 90 sec switch time, -60° angle
2. 45 sec switch time, 180° angle
3. 90 sec switch time, 60° angle
4. 90 sec switch time, -60° angle
5. 90 sec switch time, 60° angle
6. 45 sec switch time, 180° angle
7. 90 sec switch time, -60° angle
8. 90 sec switch time, 60° angle
Fig. 4 High-resolution separation with multiple states (vectors). S. cerevisiae chromosomes separated under A, two-state conditions; B, multi-state conditions. Notice separation of the co-migrating chromosomes with multi-state conditions.
Multi-state mode
20 hr run time,
120° included angle
60 to 120 sec switch-time ramp
6 V/cm (200 V), 0.5x TBE at 14°C
1.0% molecular biology Certified agarose
Secondary pulses
6 V/cm (200 V), 0° angle
3 sec switch time
4 pulses/minute
Fig. 5. Increased separation with secondary pulsed-field electrophoresis (SPFE). S. cerevisiae chromosomes separated under A, two state conditions; B, two-state conditions with secondary pulses.