Completed the remainder of the procedure for template bead titration, according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol.
Templated bead recovery after breaking emulsion, in a 1:200 dilution: 92, 105, 97, 99, 89, 96. Average = 96.3 beads/square
Calculation of beads: Avg (beads/square) x Volume in hemocytometer (uL) x total # squares on hemocytometer (squares) x dilution factor = beads/uL
Total bead recovery: beads/uL x volume of beads (uL)
So, my recovery is: 96.3 beads/square x 10uL x 25 squares x 200 = 4.817×10^6 beads/uL
Total beads recovered: 4.817×10^6 beads/uL x 200uL = 9.63×10^8 beads.
Desired # of beads is between 1-2 billion. I recovered 963 million. Close.
Final count of enriched, templated-beads: 8, 14, 16, 10. Average = 12
My final recovery is: 12 beads/square x 10uL x 25 sqaures x 10 = 30000 beads/uL.
Total enriched, templated beads recovered: 30000 beads/uL x 400uL = 12×10^6 beads.
Enrichment efficiency percentage calculation: (# templated beads)/(Starting # beads) x 100
My enrichment efficiency percentage: 12×10^6 beads/9.63×10^8 beads x 100 = 1.2%
Beads were stored @ 4C until more templated beads are generated.
Results: The yield of beads is not entirely unexpected, according to Rhonda, because I started the procedure using only 0.5pM of the library. However, each section on an octet slide (which is what we plan on using) requires 46 million beads. Clearly this is short of that quantity. The procedure will be repeated with a greater amount of starting cDNA library. It should be noted that there is NOT a linear relationship between the amount of starting template and the amount of enriched, templated beads one ends up with in this protocol. So, even though I will be increasing the starting amount of template by 3-fold, a 3-fold increase in the amount of enriched, templated beads is NOT expected (hopefully it’ll be more than that!).