Ten µl from each well is transferred to a new 384-well PCR plate to be loaded onto a MegaBACE 4000 or ABI 3730xl sequencer. Each plate is tracked with a barcode.
Our wells now contain billions of copies of our original fragment, but they are all different lengths and each ends with a dye terminator. The fragments, which have a negative charge, move through the capillaries of the sequencing machine toward the positively charged pole. Shorter fragments move faster than longer ones. At the detection window, a laser excites the dyes. A detector “reads” the colors, one at a time, to determine the sequence of the tagged As, Ts, Gs, and Cs.
Each sequencing machine can run 384 samples (lanes) through its 384 capillary tubes at one time.