Tag Archives: 3LHSITK09

SOLiD Bead Titration – Herring fragmented cDNA library 3LHSITK09 (CONTINUED from ePCR yesterday)

Completed the remainder of the procedure for template bead titration, 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.

Templated bead count after breaking emulsion, in a 1:200 dilution: 110, 129, 115, 105, 143, 113. Average = 119.2 beads/square

Recovery: 119.2 beads/square x 10uL x 25 squares x 200 = 5.96×10^6 beads/uL

Total beads recovered: 5.96×10^6 beads/uL x 200uL = 1.19×10^9

Desired # of beads is between 1-2 billion. I recovered 1.19 billion. This is in the desired range.

Final count of enriched, templated-beads: 194, 181, 161, 214. Average = 187.5 beads/square

Final recovery: 187.5 beads/square x 10uL x 25 squares x 10 = 4.6875×10^5 beads/uL

Total enriched, templated beads recovered: 4.6875×10^5 beads/uL x 400uL = 1.875×10^8 beads

Enrichment efficiency percentage: 1.875×10^8 beads/1.19×10^9 x 100 = 15.8%

Beads were stored @ 4C until more templated beads are generated.

Results: This is excellent! Got desired recovery of beads after the ePCR and got an excellent yield of templated beads. Need 46 million for a section on an octet and got 188 million, plus the 12 million from yesterdays. These two samples will be pooled. Will now proceed with the remaining herring samples, using 1.5pM as the starting amount of template for each library.

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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 Bead Titration – Herring fragmented cDNA library 3LHSITK09(CONTINUED from ePCR yesterday)

Completed the remainder of the procedure for template bead titration, according to the ABI “Templated Bead Preparation Guide” following the “full-scale” protocol.

Templated bead recovery after breaking emulsion, in a 1:200 dilution: 92, 105, 97, 99, 89, 96. Average = 96.3 beads/square

Calculation of beads: Avg (beads/square) x Volume in hemocytometer (uL) x total # squares on hemocytometer (squares) x dilution factor = beads/uL

Total bead recovery: beads/uL x volume of beads (uL)

So, my recovery is: 96.3 beads/square x 10uL x 25 squares x 200 = 4.817×10^6 beads/uL

Total beads recovered: 4.817×10^6 beads/uL x 200uL = 9.63×10^8 beads.

Desired # of beads is between 1-2 billion. I recovered 963 million. Close.

Final count of enriched, templated-beads: 8, 14, 16, 10. Average = 12

My final recovery is: 12 beads/square x 10uL x 25 sqaures x 10 = 30000 beads/uL.

Total enriched, templated beads recovered: 30000 beads/uL x 400uL = 12×10^6 beads.

Enrichment efficiency percentage calculation: (# templated beads)/(Starting # beads) x 100

My enrichment efficiency percentage: 12×10^6 beads/9.63×10^8 beads x 100 = 1.2%

Beads were stored @ 4C until more templated beads are generated.

Results: The yield of beads is not entirely unexpected, according to Rhonda, because I started the procedure using only 0.5pM of the library. However, each section on an octet slide (which is what we plan on using) requires 46 million beads. Clearly this is short of that quantity. The procedure will be repeated with a greater amount of starting cDNA library. 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 will be increasing the starting amount of 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!).

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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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