# SOLiD Bead Titration – Herring fragmented cDNA libraries: 2LHKOD09, 4LHTOG09, 6LHPWS09

Continued with templated bead prep from ePCRs for these libraries. Samples were processed 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.

2LHKOD09:

Initial counts: 109, 129, 112, 115 —- Avg. = 116.25 beads/square

Templated beads counts: 205, 199, 197, 210 —– Avg. = 210 beads/square

Templated beads: 210 x 10 x 25 x 10 = 5.06875×10^5 beads/uL x 400uL = 2.0275×10^8 beads

Efficiency: 17.44%

4LHTOG09:

Initial counts: 124, 124, 117, 104 —– Avg. = 117.25 beads/sqaure

Templated beads counts: 139, 135, 145, 140 —- Avg. = 139.75 beads/square

Templated beads: 139.75 x 10 x 25 x 10 = 3.49375×10^5 beads/uL x 400uL = 1.3975×10^8 beads

Efficiency: 11.92%

6LHPWS09:

Initial counts: 135, 106, 123, 124 —- Avg. = 122 beads/square

Templated beads counts: 141, 171, 164, 170 —– Avg. = 161.5 beads/square

Templated beads: 161.5 x 10 x 25 x 10 = 4.0375×10^5 beads/uL x 400uL = 1.615×10^8 beads

Efficiency: 13.24%

Results:

Rhonda Morales (from Ginger’s lab who is responsible for running/maintaining the SOLiD at the CEG) says the numbers on all samples look perfect! Will proceed to work flow analysis once Jesse’s samples are ready (ETA of Jan. 27th, 2010).

# SOLiD ePCRs – Herring cDNA libraries

### Herring fragmented cDNA library: 4LHTOG09

Using 1.5pM of starting template, based on success of the 3LHSITK09 bead prep (see 20100108).

Processed herring fragmented cDNA library 4LHTOG09 (20.1.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) = 201pg/uL. Mixed 89.6uL of this diluted sample with 10.4uL 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) 1/11/2010. This oil phase is stable for 2 months @ 4C.

ePCR was run. The plate will be stored @ 4C until the two remaining libraries have been through ePCR. Then, all three libraries will be processed simulatneously.

### Herring fragmented cDNA library: 6LHPWS09

Using 1.5pM of starting template, based on success of the 3LHSITK09 bead prep (see 20100108).

Processed herring fragmented cDNA library 6LHPWS09 (51.4.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) = 541pg/uL. Mixed 35uL of this diluted sample with 65uL 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) 1/11/2010. This oil phase is stable for 2 months @ 4C.

ePCR was run. The plate will be stored @ 4C until the two remaining libraries have been through ePCR. Then, all three libraries will be processed simultaneously.