Tag Archives: bead counts

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 Templated Bead Prep – Yellow perch CT, WB and lake trout Lean libraries (continued from yesterday)

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 counts: (1:200 dilution)

CT: 132, 133, 127, 136 Avg. = 132

WB: 127, 128, 119, 126 Avg. = 125

Lean: 121, 114, 132, 109 Avg. = 119

CT: 132 x 25 x 10 x 200 = 6.6×10^6 beads/uL x 200uL = 1.32×10^9 beads

WB: 125 x 25 x 10 x 200 = 6.25×10^6 beads/uL x 200uL = 1.25×10^9 beads

Lean: 119 x 25 x 10 x 200 = 5.95×10^6 beads/uL x 200uL = 1.19×10^9 beads

Templated Bead counts (1:10 dilution)

CT: 91, 80, 100, 78 Avg. = 87.25

WB: 69, 70, 75, 65 Avg. = 69.75

Lean: 40, 52, 48, 46 Avg. = 46.5

CT: 87.25 x 25 x 10 x 10 = 218125 beads/uL x 400uL = 8.7525×10^7 beads

WB: 39.75 x 25 x 10 x 10 = 174375 beads/uL x 400uL = 6.975×10^7 beads

Lean: 46.5 x 25 x 10 x 10 = 116250 beads/uL x 400uL = 4.65×10^7 beads

Percent Recovery Templated Beads

CT: (8.7252×10^7 beads)/(1.32×10^9 beads) x 100 = 6.61%

WB: (6.975×10^7 beads)/(1.25×10^9 beads) x 100 = 5.58%

Lean: (4.65×10^7 beads)/(1.19×10^9 beads) x 100 = 3.91%

Results: Everything looks pretty darn good. One mild concern, however, is the yield from the the Lean library. An 8-well slide requires 41 million beads for a run. Additionally, I believe 15 million are needed for a WFA (quality check, pre-run). This means that the Lean prep is nearly 10 million beads short of what is necessary for a “complete” run of this sample. Will send the numbers to Rhonda and see what her opinion is and what she suggests to do. But, based on the percent recovery, all the samples should be really high quality (extremely few polyclonal beads).

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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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Templated Bead Prep SOLiD Libraries – Abalone CC, CE pools and yellow perch CT, PQ 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

CC: 127, 120, 126, 113. Average = 96 Count: 96 x 10 x 25 x 200 x 200 = 9.6 x 10^8 beads

CE: 99, 93, 115, 102. Average = 102.25 Count: 102.25 x 10 x 25 x 200 x 200 = 1.0225 x 10^9 beads

CT: 118, 113, 109, 111. Average = 112.75 Count: 112.75 x 10 x 25 x 200 x 200 = 1.1275 x 10^9 beads

PQ: 109, 116, 111, 86. Average = 105.5 Count: 105.5 x 10 x 25 x 200 x 200 = 1.055 x 10^9 beads

Templated Bead Counts

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

CC: 217, 226, 208, 219 Average = 217.5 Count: 217.5 x 10 x 25 x 10 x 400 = 2.175 x 10^8 beads

CE: 211, 169, 162, 180 Average = 180.5 Count: 180.5 x 10 x 25 x 10 x 400 = 1.805 x 10^8 beads

CT: 223, 219, 254, 214 Average = 227.5 Count: 227.5 x 10 x 25 x 10 x 400 = 2.275 x 10^8 beads

PQ: 176, 177, 161, 163 Average = 169.25 Count: 169.25 x 10 x 25 x 10 x 400 = 1.6925 x 10^8 beads

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

CC = 2.17 x 10^8 beads/9.6 x 10^8 beads x 100 = 22.7%

CE = 1.805 x10^8 beads/10.225 x 10^8 beads x 100 = 17.7%

CT = 2.275 x 10^8 beads/11.275 x 10^8 beads x 100 = 20.2%

PQ = 1.6925 x 10^8 beads/10.55 x 10^8 beads x 100 = 16.04%

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 CC and CT 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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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 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 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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