The presence of ions can interfere with the uptake of DNA using electrokinetic injection because of preferential injection of higher charge-to-mass molecules (e.g. Cl¯ ions). Therefore it is essential to desalt the inactivated restriction digest with Microcon columns (Amicon), Qiaquick Nucleotide Removal Kit (Qiagen) or conventional ethanol precipitation. In our case, the restriction products were diluted with water up to 500 μl, before concentration and desalinization on Microcon columns (see figure below).
6) Capillary electrophoresis (CE); loading. A fifth to a third of the desalted restriction digest is generally loaded onto the capillary electrophoresis system using the default settings optimized for sequencing. This can lead to insufficient fluorescent signal, therefore yielding a low number and height of peaks. The injection time and injection voltage can be varied to regulate DNA uptake. The default sample injection time is generally 10 sec, however longer injection times increase the uptake of DNA from dilute sample solutions, therefore yielding a higher fluorescent signal with more peaks detected. Furthermore, injection voltage is directly proportional to the amount of DNA injected and can be modified from the default of 3 kV. Further concentration of the desalted product may also be necessary to stay within the appropriate CE loading volume.
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