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 -
Rec’d package from Rutgers (Emily Pearson) containing two large Ziplock bags on “wet” ice, each of those containing smaller bags with sample tubes in them. One large bag contains gill tissue samples and the other large bag contains hemolymph samples. Samples will temporarily be stored @ 4C until they can be catalogued and boxed by Lexie later today.
Note: This information was added 20140407. Yes, you read that correctly.
Someone had noticed that gDNA isolated using a Qiagen DNeasy Blood & Tissue Kit we rec’d in April 2010 seemed to be yielding degraded DNA.
The two samples used for the comparison were a single tail (split in two equal weight pieces) from a juvenile salmon that was snap frozen, without preservatives, at the time of its collection. The samples were prepped. 0.5ug of eluted DNA was then run on a 1.2% agarose-TAE gel containing 0.1ug/mL of ethidium bromide. 5uL of Bioline’s Hyperladder I was loaded for size assessment (see link for marker layout).
Results:
Clearly, there’s significant quality difference! A free replacement kit was sent by Qiagen.
Utilized to sets of primers obtained from the Friedman Lab: VptA (referred to as “Hasegawa”, even though the reference article calls the primers Vtp A) and Vt IGS (referred to as “Lee” primers, presumably from a published article). For template, used “RE22 DNA” that was given to me by Elene. Tube is dated 9/10/09 and has no indication of concentration. Performed qPCR on a set of 10-fold dilutions. Plate layout/qPCR set up is here, along with dilution series used.
Results:
The VptA primer set generated a nice looking set of dilutions with appropriate spacing (~3.2 Ct/10-fold dilution). HOWEVER, the raw fluorescence signal is very low (only 0.4 units; good signal is usually 3-5-fold higher) AND the melting curve doesn’t look that great. The melting curve could look poor due to the low signal, since it doesn’t come up much higher than background levels.
It should be noted that the low fluorescence levels generated could simply be due to the amplicon size generated by these primers. The amplicon size is only 63bp. An amplicon of this size might not be able to incorporate significant amounts of dye to generate a “normal” level of fluorescence (1.25 – 2 units).
The Vt IGS primers failed to generate any product.
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.
