What is the easiest and most cost efficient means to remove the Dye Deoxy-terminators for automated sequencing after cycle sequencing?
The vast majority of fluorescent ddNTPs are not incorporated in the PCR products of cycle sequencing . If they are not removed effectively they form an enormous peak at the start of a run and cause streaking artifacts for several hundred bases afterwards, thus seriously degrading the quality of the sequence. The best way to remove the unincorporated dyes is to use a sephadex G-50 spin column. Make a hole in the bottom of 0.5 ml eppendorf tube using a hot 30 gauge needle and add about 25 ul volume of silanized zirconium glass beads. Pour sephadex G-50 in 0.3 M sodium acetate (5 grams in 60 ml) to the top of the tube. Place the small eppendorf tube inside a larger 1.5 ml eppendorf tube and spin at about 500 rpm for couple of minutes to remove the excess liquid from the matrix. Transfer the smaller tube to a clean siliconized 1.5 ml tube. The DNA sample is added on top of the sephadex matrix and again spun at 1500 RPM for 2 minutes. The eluate is either precipitated by at least 3 volumes
