Repeated PCR from 20130919, but with a newly designed reverse primer. Primers were designed using MethPrimer (SR IDs: 1553, 1555). Primers were designed using MethPrimer:
Results:
No amplification of any samples BS-treated or non-treated.
Repeated PCR from 20130919, but with a newly designed reverse primer. Primers were designed using MethPrimer (SR IDs: 1553, 1555). Primers were designed using MethPrimer:
Results:
No amplification of any samples BS-treated or non-treated.
Repeated PCR from 20130919, but with a newly designed set of primers that targets the desired region of C1q for sequencing (SR IDs: 1553, 1554). Primers were designed in Geneious.
Results:
No amplification of any samples BS-treated or non-treated.
Ran PCR on lake trout DNA and lake trout bisulfite-converted DNA. Used primers SRIDs: 1551 and 1552. DNA was isolated by Caroline Storer on 4/4/2011 and bisulfite converted on 4/7/2011. Master mix and cycling params are here:
http://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20130919-01.jpg
Samples:
Lake Trout Lean_6 Liver
Lake Trout Lean_7 Liver
Lake Trout Siscowet_6 Liver
Lake Trout Siscowet_7 Liver
Bisulfite converted DNA from the four samples listed above.
Results:
Lane 1: Hyperladder II (Bioline)
Lane 2: Lean6
Lane 3: Lean6 BS
Lane 4: Lean7
Lane 5: Lean7 BS
Lane 6: Siscowet6
Lane 7: Siscowet6 BS
Lane 8: Siscowet7
Lane 9: Siscowet7 BS
All the non-BS converted samples amplified as expected, producing a band of ~560bp. However, none of the BS-converted DNA produced any amplification. It is likely an issue with the primer sequences and the resulting conversion of the gDNA.
Will look at Caroline Storer’s notebook entries for her work on this and try to evaluate what has already been done.
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.
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).
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
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
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.