Separation

Separation

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The cathode buffer reservoir, a water reservoir, and a waste vial are located in the autosampler. A short period of electrophoresis injects the sample into the capillary. Next, the autosampler moves the cathode buffer reservoir to the capillary and cathode electrode to continue electrophoresis. The buffer used in the CE system is viscous and adheres to the exterior of the capillary during the buffer fill step. In the subsequent injection step this material could contaminate the sample, change its ionic strength, and reduce the quantity of material injected. The water reservoir is used to wash the cathode and capillary tip between sample injections.

The buffer solution moves through the capillary under the influence of an electric field. This phenomenon is termed electroosmotic or electroendosmotic flow. The direction of the electroosmotic flow is toward the positively charged anode, which means that the buffer flows from the source vial, through the capillary, through the detector, to the destination vial. The DNA fragments migrate towards the anode reservoir.

Performance Optimized Polymers™ dynamically coat the capillary wall to control electroosmotic flow during electrophoresis. The most common polymer used is ABI’s Performance Optimized Polymer™-4 (POP-4™).07 This polymer was developed to meet the following specifications:

detect alleles differing in size by a single base (up to 250 base pairs in length)
size alleles of the same length with a precision of less than 0.15 nucleotide standard deviation
require less than 30 minutes analysis time per sample
provide capillary life of at least 100 injections
provide a highly denaturing environment for the DNA samples07

Maintaining a Capillary

The capillary must be properly maintained in order to achieve reproducible separation of DNA fragments. Capillaries must not be allowed to dry. If the capillary inlet and outlet dry out, then urea or salts from the buffer will form crystals that clog that narrow openings. A clogged capillary can result in low or no current when voltage is applied. The end of capillaries should be stored in deionized water or buffer to prevent drying out.

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