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Time (min)

Figure 7.5 (A) Separation of Hae III digested <£X174 RF DNA, 50 /ug/mL in 20 mM NaCl. (B) Influence of presample injection of 0.1 M Tris-acetate, pH 8.3. First injection: 10-second pressure injection of Tris-acetate; second injection: 20-second injection of digest. [Reprinted with permission from van der Schans et al., / Chromatogr A 680:511 (1994).]

is now no longer a linear function. To correct this, a double injection technique is employed, whereby an unknown and a standard are injected sequentially and allowed to comigrate in the capillary. Migration times for the coinjected marker fragments generate the molecular size curve, which is then used to determine the relative size of the unknown DNA fragment. Order of injection is important (van der Schans et al., 1994)—since salt from the PCR sample will migrate faster than the DNA components, it can create a leading zone of low field strength. An injection order of sample followed by standard will cause fronting of the PCR product, but sharp peaks for the standard. The opposite is seen for an injection order of standard followed by sample. This effect is demonstrated in Figure 7.6. Alternatively, an internal standard may be included in the sample (e.g., PCR coamplification of a second target sequence, addition of a known quantity of DNA to each sample).

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"t 12

Time (min)

Time (min)

Figure 7.6 (A) Electropherogram of PCR sample, then DNA standard. First injection: PCR sample (97 bp); second injection: Hae Ill-digested 0X174 RF DNA, 10 ¿tg/mL. (B) Electropherogram of DNA standard, then PCR sample. First injection: Hae Ill-digested 0X174 RF DNA, 10 ¿xg/mL; second injection: PCR sample (97 bp). [Reprinted with permission from van der Schans et al., J Chromatogr A 680:511 (1994).]

Time (min)

Figure 7.6 (A) Electropherogram of PCR sample, then DNA standard. First injection: PCR sample (97 bp); second injection: Hae Ill-digested 0X174 RF DNA, 10 ¿tg/mL. (B) Electropherogram of DNA standard, then PCR sample. First injection: Hae Ill-digested 0X174 RF DNA, 10 ¿xg/mL; second injection: PCR sample (97 bp). [Reprinted with permission from van der Schans et al., J Chromatogr A 680:511 (1994).]

7.3.8 Artifacts

An injection-related artifact can occur in gel buffers with consecutive electroki-netic injections from the same, low volume (10-200 mL) sample: progressively smaller amounts of sample are introduced into the capillary, resulting in peak heights or areas that decrease with each injection (Schwartz et al., 1995). This effect is due to the migration of cations (e.g., Tris) from the gel buffer into the sample, changing its relative ionic strength. One solution to this problem is to perform an electrokinetic injection from a water vial prior to sample injection. This water injection generates a zone of "ion depletion" (i.e., rela-

tively high resistance and low conductivity) at the cathode end of the capillary. As the PCR sample is subsequently electrokinetically injected into the capillary, very few cations will migrate into the sample vial from the capillary. Hence, the sample solution will stay relatively salt-free, even with subsequent injections from the same vial. This two-step injection procedure not only results in dramatically increased precision (important for quantitative studies), but also increases sample loading. Because a relatively high resistance, low conductivity zone has been introduced into the capillary, the local field strength in this zone is relatively high—therefore, more sample is pulled into the capillary than would have made its way there without the preinjection of water.

A second injection-related artifact can occur in physical gel networks: poor peak shape (i.e., severe tailing), with capillaries improperly cut at the inlet side. An oblique shape at the injection site of the capillary yields a separation efficiency significantly lower than that obtainable with a properly cut, straightedge capillary (Schwartz et al., 1995). When these jagged-edge capillaries are used, the injection plug length is slightly increased (relative to the straightedge capillary), resulting in increased extracolumn variance due to the injection plug. Care must be taken to ensure that the capillary ends are properly prepared and maintained.

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