Tag Archives: ovary

Templated Bead Prep SOLiD Libraries – Yellow perch WB, lake trout Lean and Sisco, and herring G/O HWS09 libraries

All libraries were prepped according to ABI’s “full-scale” bead prep protocol. Initial bead counts were performed using a hemocytometer in a 1:200 dilution:

Formula for calculating bead counts:

Average hemo count x hemo volume x hemo squares x dilution x bead volume

Initial Bead Counts

WB: 111, 96, 90, 100 Average = 99.25 Count: 99.25 x 10 x 25 x 200 x 200 = 9.925 x 10^8 beads

Lean: 101, 100, 108, 108 Average = 104.25 Count: 104.25 x 10 x 25 x 200 x 200 = 1.0425 x 10^9 beads

Sisco: 142, 144, 120, 112 Average = 129.5 Count: 129.5 x 10 x 25 x 200 x 200 = 1.295 x 10^9 beads

HPWS09: 112, 115, 105, 104 Average = 109 Count: 109 x 10 x 25 x 200 x 200 = 1.09 x 10^9 beads

Templated Bead Counts

Templated bead counts were performed using a hemocytometer with a 1:10 dilution:

WB: 198, 186, 198, 175 Average = 189.25 Count: 189.25 x 10 x 25 x 10 x 400 = 1.8925 x 10^8 beads

Lean: 253, 259, 236, 244 Average = 248 Count: 248 x 10 x 25 x 10 x 400 = 2.48 x 10^8 beads

Sisco: 267, 241, 252, 255 Average = 253.75 Count: 253.75 x 10 x 25 x 10 x 400 = 2.5375 x 10^8 beads

HPWS09: 193, 193, 172, 186 Average = 186 Count: 186 x 10 x 25 x 10 x 400 = 1.86 x 10^8 beads

Templated Bead Recovery: Final bead count divided by initial bead count x 100 = % recovery

WB = 1.8925 x 10^8/9.925 x 10^8 x 100 = 19.07%

Lean = 2.48 x 10^8/1.0425 x 10^9 x 100 = 23.8%

Sisco = 2.5375 x 10^8/1.295 x 10^9 x 100 = 24.34%

HPWS09 = 1.86 x 10^8/1.09 x 10^9 x 100 = 17.06%

Results: Yields of templated beads look fabulous. Recoveries of templated beads are a bit on the high side (desired recoveries are between 5-15%, with 20% being the “cutoff” that Rhonda’s lab uses for runs. The Lean and Sisco samples cross this cutoff value. Will consult with Steven to see what how he wants to proceed (i.e. new ePCRs?). Beads stored @ 4C until ready for running on the SOLiD.

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Herring 454 Data

Data from MoGene was received today on two DVDs and one HDD. Data is two runs of two libraries, due to MoGene concerns that the data of the first run looked bad (too few reads). They performed a second run at no charge and provided us with that data as well.

 

UPDATE 20150310

Data is located here: http://owl.fish.washington.edu/nightingales/C_pallasii/

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mRNA Isolation – Herring gonad/ovary RNA (from 20091023)

RNA Precipitation

Sample was spun 16,000g, 30mins, 4C. Supe removed. Pellet washed with 1mL 70% EtOH. Spun 16,000g, 10mins, 4C. Supe removed. Pellet resuspended in 250uL of nuclease free H2O. Will proceed with mRNA isolation.

 

mRNA Isolation

Isolated mRNA using Ambion’s MicroPolyA Purist Kit according to protocol. Performed two rounds of isolation to decrease residual rRNA carryover that we frequently see after a single round.

Results:

Started with ~90ug of total RNA. Yield of mRNA = 3.26ug. That is a ~3.6% recovery of mRNA.

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RNA Precipitation – Herring gonad/ovary RNA (from 20091023)

A subset (3 samples from each group) of samples were pooled (see spreadsheet, green-highlighted samples), each providing ~7.5ug of RNA, yielding 112.17uL. 0.1 vols of 3M NaOAC, pH = 5.2 were added to the tube (11.22uL). 2 vols of EtOH (246.8uL) was added to the tube. Tube was vortexed to mix and incubated @ -20C O/N.

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RNA Isolation – Herring Gonad/Ovary Samples

RNA was isolated according to protocol. Pellets were resuspended in 50uL of 0.1%DEPC-H2O, heated @ 55C for 5 mins, spec’d and stored @ -80C in the “Herring RNA Box #1″.

Results:

Most of the samples look good, however there are a number of samples that are downright bad. Either no RNA or very low concentrations with poor 260/280, 260/230 ratios.

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RNA Isolation – Herring Gonad/Ovary Samples

From the Seeb Lab. Homogenized entire gonad/ovary samples in 5mL of TriReagent with the sonicator. In essence, based on the manufacturer’s recommendation, this means the ratio of tissue:TriReagent was ~2x. Transferred 0.5mL of homogenized gonad/ovary sample to 1.5mL snap cap tubes and added an additional 0.5mL of TriReagent, to adjust the ratio of tissue:TriReagent to ~1x. Samples were then stored @ -80C. These will be further processed tomorrow.

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