Completed the remainder of the procedure for template bead titration, according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol.
To see explanations of the various calculations below, see the “SOLiD Bead Titration – Herring fragmented cDNA library 3LHSITK09(CONTINUED from ePCR yesterday)” from 20100107.
Templated bead count after breaking emulsion, in a 1:200 dilution: 110, 129, 115, 105, 143, 113. Average = 119.2 beads/square
Recovery: 119.2 beads/square x 10uL x 25 squares x 200 = 5.96×10^6 beads/uL
Total beads recovered: 5.96×10^6 beads/uL x 200uL = 1.19×10^9
Desired # of beads is between 1-2 billion. I recovered 1.19 billion. This is in the desired range.
Final count of enriched, templated-beads: 194, 181, 161, 214. Average = 187.5 beads/square
Final recovery: 187.5 beads/square x 10uL x 25 squares x 10 = 4.6875×10^5 beads/uL
Total enriched, templated beads recovered: 4.6875×10^5 beads/uL x 400uL = 1.875×10^8 beads
Enrichment efficiency percentage: 1.875×10^8 beads/1.19×10^9 x 100 = 15.8%
Beads were stored @ 4C until more templated beads are generated.
Results: This is excellent! Got desired recovery of beads after the ePCR and got an excellent yield of templated beads. Need 46 million for a section on an octet and got 188 million, plus the 12 million from yesterdays. These two samples will be pooled. Will now proceed with the remaining herring samples, using 1.5pM as the starting amount of template for each library.