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 -
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.
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
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.
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
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
Amplified cDNA was cleaned up using the Invitrogen PureLink Micro Kit, but was done so according to Ambion’s Whole Transcriptome Analysis Kit protocol and then spec’d.
Results:
0.5uL was removed from each sample and mixed with 0.5uL to run on DNA 1000 chips on the Bioanalyzer 2100. The slideshow below shows the electropherograms from each sample. Each sample (to be considered worthy of moving to the next stage) should have <20% of the sample in the 25-150bp range. All 8 samples exhibit this and their peaks look very good. Will proceed to ePCR/templated bead prep next week.
cDNA was gel purified according to Ambion’s Whole Transcriptome Analysis Kit. The appropriate regions (100 – 200bp) were excised and cut in to 4, 1x5mm pieces. The two “internal” pieces were transferred to individual PCR tubes. The “outer” pieces were transferred together to a 1.5mL snap cap tube and stored @ -20C.
Three images are below. The first two are the gels before excising the 100 – 200bp region of the gel. The third is the image of the SECOND gel after the specified region was excised. An image was not taken of Gel 1 after excision (whoops!).
Gel 1
Gel 2
NOTE: The WB sample in the gell above is actually a yellow perch sample, NOT an abalone sample!
Gel 2 AFTER EXCISION
NOTE: The WB sample in the gell above is actually a yellow perch sample, NOT an abalone sample!
In-gel PCR was performed on the individual “internal” gel pieces that were excised, as described below from earlier today. PCR rxns/cycling were performed according to Ambion’s Whole Transcriptome Analysis Kit. PCR ran O/N. PCR master mix set up is here (bottom half of sheet).
Samples were speedvac’d to dryness and resuspended in 3uL of nuclease-free H2O. Samples were then reversed transcribed according to Ambion’s Whole Transcriptome Analysis Kit. RT master mix set up is here (top portion of sheet).
All 8 samples were hybridized/ligated according to Ambion’s Whole Transcriptome Analysis Kit using Adaptor A.