All libraries were prepped according to ABI’s “full-scale” bead prep protocol. Initial bead counts were performed using a hemocytometer in a 1:200 dilution:

Formula for calculating bead counts:

Average hemo count x hemo volume x hemo squares x dilution x bead volume

Initial Bead Counts

CC: 127, 120, 126, 113. Average = 96 Count: 96 x 10 x 25 x 200 x 200 = 9.6 x 10^8 beads

CE: 99, 93, 115, 102. Average = 102.25 Count: 102.25 x 10 x 25 x 200 x 200 = 1.0225 x 10^9 beads

CT: 118, 113, 109, 111. Average = 112.75 Count: 112.75 x 10 x 25 x 200 x 200 = 1.1275 x 10^9 beads

PQ: 109, 116, 111, 86. Average = 105.5 Count: 105.5 x 10 x 25 x 200 x 200 = 1.055 x 10^9 beads

Templated Bead Counts

Templated bead counts were performed using a hemocytometer with a 1:10 dilution:

CC: 217, 226, 208, 219 Average = 217.5 Count: 217.5 x 10 x 25 x 10 x 400 = 2.175 x 10^8 beads

CE: 211, 169, 162, 180 Average = 180.5 Count: 180.5 x 10 x 25 x 10 x 400 = 1.805 x 10^8 beads

CT: 223, 219, 254, 214 Average = 227.5 Count: 227.5 x 10 x 25 x 10 x 400 = 2.275 x 10^8 beads

PQ: 176, 177, 161, 163 Average = 169.25 Count: 169.25 x 10 x 25 x 10 x 400 = 1.6925 x 10^8 beads

Templated Bead Recovery: Final bead count divided by initial bead count x 100 = % recovery

CC = 2.17 x 10^8 beads/9.6 x 10^8 beads x 100 = 22.7%

CE = 1.805 x10^8 beads/10.225 x 10^8 beads x 100 = 17.7%

CT = 2.275 x 10^8 beads/11.275 x 10^8 beads x 100 = 20.2%

PQ = 1.6925 x 10^8 beads/10.55 x 10^8 beads x 100 = 16.04%

Results: Yields of templated beads look fabulous. Recoveries of templated beads are a bit on the high side (desired recoveries are between 5-15%, with 20% being the “cutoff” that Rhonda’s lab uses for runs. The CC and CT samples cross this cutoff value. Will consult with Steven to see what how he wants to proceed (i.e. new ePCRs?). Beads stored @ 4C until ready for running on the SOLiD.