Tag Archives: 4LHTOG09

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

Beads: 116.25 x 10 x 25 x 200 = 5.8125×10^6 beads/uL x 200uL = 1.1625×10^9 beads

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

Beads: 117.25 x 10 x 25 x 200 = 5.8625×10^6 beads/uL x 200uL = 1.1725×10^9 beads

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

Beads: 122 x 10 x 25 x 200 = 6.1×10^6 beads/uL x 200uL = 1.22×10^9 beads

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%

All beads were stored @ 4C until ready for bead deposition and work flow analysis run.

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).

Here are reagent lot numbers for the Bead Titration.

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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.

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