Tag Archives: ePCR

SOLiD ePCR/Templated Bead Prep – Lake Trout Lean library

ePCR was performed following ABI’s “full scale” protocol, using 1pM of SOLiD cDNA library.

Templated bead preparation was performed according to the “full scale” protocol.

Bead counts are calculated as follows:

Avg bead count x # hemacytometer squares x volume in hemacytometer (uL) x dilution factor = beads/uL x suspension volume (uL) = total beads

Initial Bead count: (1:200 dilution)

Lean: 126, 138, 122, 138 Avg. = 131

Lean: 131 x 25 x 10 x 200 = 6.55×10^6 beads/uL x 200uL = 1.31×10^9 beads

Templated Bead counts (1:10 dilution)

Lean: 165, 171, 186, 160 Avg. = 170.5

170.5 x 25 x 10 x 10 = 426250 beads/uL x 400uL = 1.705×10^8 beads

Percent Recovery Templated Beads

Lean: (1.705×10^8 beads)/(1.31×10^9 beads) x 100 = 13.02% recovery

Results: Yield is significantly higher than the previous preparation performed with this sample. The percent recovery falls into the desired range of 5-15%, so things look good there, too. Will contact Rhonda and get info regarding when this and the other 7 samples can go on a run.

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SOLiD ePCRs – Yellow perch CT, WB and lake trout Lean libraries

Performed ePCRs on these samples from DATE, following the “full scale” protocol. A work flow analysis (WFA) run on these samples from the initial ePCRs/templated bead prep (DATE) revealed too many polyclonal beads, thus requiring them to be redone. ePCRs will be performed using 1.0pM (120pg/uL) of the SOLiD cDNA fragment libraries, instead of the 1.5pM (180pg/uL) used previously.

CT –

WB –

Lean -

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ePCR SOLiD Libraries – Lake Trout Sisco and Herring G/O HPWS09 libraries (from 20100408)

ePCR was performed for the above three mentioned SOLiD libraries using 1.5pM (180 pg/uL) of cDNA, according to the ABI “full scale” ePCR protocol. ePCRs were stored @ 4C until ready for the emulsion breaking step.

Amounts of cDNA used to make dilutions (in 1x Low TE Buffer) of 180pg/uL:

Sisco (42.29 ng/uL): 2.13uL in 500uL

HPWS09 (9.29 ng/uL): 1.94uL in 100uL

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ePCR SOLiD Libraries – Yellow perch PQ, WB and Lake Trout Lean libraries (from 20100408)

ePCR was performed for the above three mentioned SOLiD libraries using 1.5pM (180 pg/uL) of cDNA, according to the ABI “full scale” ePCR protocol. ePCRs were stored @ 4C until ready for the emulsion breaking step.

Amounts of cDNA used to make dilutions (in 1x Low TE Buffer) of 180pg/uL:

PQ (20.77ng/uL): 4.33uL in 500uL

WB (16.81ng/uL): 2.14uL in 200uL

Lean (53.49ng/uL): 1.68uL in 500uL

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ePCR SOLiD Libraries – Abalone CC, CE pools and yellow perch CT SOLiD libraries (from 20100408)

Emulsion PCR (ePCR) was performed for the above three mentioned SOLiD libraries using 1.5pM (180 pg/uL) of cDNA, according to the ABI “full scale” ePCR protocol. ePCRs were stored @ 4C until ready for the emulsion breaking step.

Amounts of cDNA used to make dilutions (in 1x Low TE Buffer) of 180pg/uL:

CC (13.8ng/uL): 1.3uL in 100uL

CE (23.01ng/uL): 1.56uL in 200uL

CT (63.8ng/uL): 1.41 in 500uL

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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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SOLiD ePCR – Herring fragmented cDNA library: 2LHKOD09

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

Processed herring fragmented cDNA library 2LHKOD09 (76.1.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) = 761pg/uL. Mixed 23.7uL of this diluted sample with 76.3uL 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 started and run O/N. The plate will be stored @ 4C until the two remaining libraries have been through ePCR. Then, all three libraries will be processed simulatneously.

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SOLiD ePCR – Herring fragmented cDNA library: 3LHSITK09

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.

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SOLiD ePCR – Herring fragmented cDNA library: 3LHSITK09 (from 20091209)

Processed herring fragmented cDNA library 3LHSITK09 (88.ng/uL) according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol. Made a 1:1000 dilution (1uL library, 999uL 1x Low TE) = 88.5pg/uL. Mixed 67.8uL of this diluted sample with 32.2uL 1x Low TE to get a final concentration of 60pg/uL (500pM, 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 (Rhonda will put plate in fridge for storage O/N) and the rest of the procedure will be finished tomorrow.

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Emulsion PCR – Herring Liver cDNA for SOLiD Libraries

Emulsion PCR was performed with the two inner gel bands cut out earlier today according to the Ambio WTK protocol. PCR was run for 15 cycles. After the PCR, the samples were cleaned up using the Invitrogen PureLink PCR Micro Kit, according to the Ambion WTK protocol. The cleaned up cDNA (referred to as “libraries” from now on) was spec’d prior to running on the Bioanalyzer.

Results:

All samples look good EXCEPT for 4L. It has a terrible 260/230 ratio and has a very low concentration, relative to the other three samples. Although not pictured here, the absorbance curve of the 4L sample was extremely poor and broad, unlike the other three samples.

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