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
WB: 111, 96, 90, 100 Average = 99.25 Count: 99.25 x 10 x 25 x 200 x 200 = 9.925 x 10^8 beads
Lean: 101, 100, 108, 108 Average = 104.25 Count: 104.25 x 10 x 25 x 200 x 200 = 1.0425 x 10^9 beads
Sisco: 142, 144, 120, 112 Average = 129.5 Count: 129.5 x 10 x 25 x 200 x 200 = 1.295 x 10^9 beads
HPWS09: 112, 115, 105, 104 Average = 109 Count: 109 x 10 x 25 x 200 x 200 = 1.09 x 10^9 beads
Templated Bead Counts
Templated bead counts were performed using a hemocytometer with a 1:10 dilution:
WB: 198, 186, 198, 175 Average = 189.25 Count: 189.25 x 10 x 25 x 10 x 400 = 1.8925 x 10^8 beads
Lean: 253, 259, 236, 244 Average = 248 Count: 248 x 10 x 25 x 10 x 400 = 2.48 x 10^8 beads
Sisco: 267, 241, 252, 255 Average = 253.75 Count: 253.75 x 10 x 25 x 10 x 400 = 2.5375 x 10^8 beads
HPWS09: 193, 193, 172, 186 Average = 186 Count: 186 x 10 x 25 x 10 x 400 = 1.86 x 10^8 beads
Templated Bead Recovery: Final bead count divided by initial bead count x 100 = % recovery
WB = 1.8925 x 10^8/9.925 x 10^8 x 100 = 19.07%
Lean = 2.48 x 10^8/1.0425 x 10^9 x 100 = 23.8%
Sisco = 2.5375 x 10^8/1.295 x 10^9 x 100 = 24.34%
HPWS09 = 1.86 x 10^8/1.09 x 10^9 x 100 = 17.06%
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 Lean and Sisco 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.