美國Bio-rad伯樂CHEF Mapper XA脈沖場電泳儀
CHEF MAPPER XA 是目前的脈沖場電泳系統(tǒng)，擁有設(shè)計的六角形電極的電泳槽，保證矢量電場的自由旋轉(zhuǎn)，比常規(guī)的脈沖場電泳提供更高的分辨率，電泳速度和更精確的分離，對100bp-10Mb的DNA片段都能提供有效的分離。主要用于基因組DNA的分離分析，大分子DNA的指紋圖譜分析及多態(tài)性分析等。
1．自動演算：Bio-Rad積多年在脈沖場電泳方面的經(jīng)驗，提供給用戶一套程序用于優(yōu)化實驗參數(shù)。只要輸入待分離DNA片段的最小、長度，結(jié)合10個主要變量的確定，幫助使用者獲得的實驗條件。
2．脈沖角度：可以在0-360°間自由選擇脈沖角度，使用戶可以在同一系統(tǒng)上實現(xiàn)大至染色體級、小至質(zhì)粒DNA的有效分離。
3．時間轉(zhuǎn)換梯度：有線性、非線性（凸形和凹形）兩種脈沖時間梯度，非線性梯度可以提供更廣泛的分離動態(tài)范圍，使用戶可以精確的確定分離片段的大小。
4．多狀態(tài)功能：CHEF Mapper XA在一個Block中可以有15個電場矢量，每個電場矢量可以有自己的電壓和轉(zhuǎn)換時間，可有選擇地對一定大小范圍的片段進行更精細的分離，并且可以在同一次電泳中實現(xiàn)FIGE和CHEF兩種技術(shù)。
5．二次脈沖功能：二次脈沖可加速DNA從瓊脂糖凝膠中釋放，從而有利于非常大的DNA片段的分離，并可提高分辨率。
6．技術(shù)應(yīng)用：
CHEF（鉗式均衡電場）技術(shù)，產(chǎn)生均衡電場；
PACE（程序自主控制電極）技術(shù)，根據(jù)片斷大小的需要優(yōu)化設(shè)定脈沖角度；
FIGE（電場倒置）技術(shù)，為 250KB以下小片斷DNA提供快速分離；
AFIGE（非對稱場倒置）技術(shù)，精細分離小片斷DNA，提高分辨率；
以上技術(shù)的應(yīng)用保證了科研人員在所有分子量范圍內(nèi)均能得到所的線性分離。
性能指標：
a. 電源輸出：電壓 350V，0和0.6-9V/cm，0.1V/cm增量，連續(xù)可調(diào)
b. 電流：0.5A
c. 延遲啟動：72小時
d. 電極調(diào)節(jié)能力：動態(tài)調(diào)節(jié)（反饋調(diào)整）±0.5％
e. 程序儲存：存儲20個復(fù)雜實驗程序，每個程序包含8個程序模塊或99個簡單程序
f. 數(shù)據(jù)記錄：鍵盤，條形碼讀取或RS-232接口
g. 顯示屏：2行×40字符/行，熒光顯示
h. 轉(zhuǎn)換范圍：50毫秒到18小時
i. 轉(zhuǎn)換角度：0-360°，0.5°增量
j. 電泳時間：999小時/模塊
脈沖中斷設(shè)置：可以通過電壓，頻率，角度和持續(xù)時間設(shè)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.
