Due to low yield of templated beads (12×10^6) from the first run through (see SOLiD Bead Titration below), am repeating using 1.5pM of starting template (as opposed to 0.5pM used yesterday). 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 am increasing the starting 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!).
Processed herring fragmented cDNA library 3LHSITK09 (88.ng/uL) according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol. Made a 1:100 dilution (1uL library, 99uL 1x Low TE) = 885pg/uL. Mixed 20.3uL of this diluted sample with 79.7uL 1x Low TE to get a final concentration of 180pg/uL (1.5pM, according to ABI protocol). Oil phase used was previously prepared by Jesse (Seeb lab) in mid-December 2009. This oil phase is stable for 2 months @ 4C.
ePCR was started and run O/N. The rest of the procedure will be finished tomorrow.