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Rui:LabNotes/Haplotyping/2014-11-12
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==Oligo set 90k_oligos_30Sept2014== * Received on 11.12.14 * Kun's design note: http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/MONOD/2014-8-4#Re-design_of_selector_probes * Noi's latest handling note: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-10-15 ===Basic information=== *Sept14 probe set: [[Media:90k_oligos_30Sept2014.txt.gz]] probe set # probes Length Amp primers Chris gDNA_MS_v2 8,045 127bp V6(G*T*CATATCGGTCACTGTU//5Phos/GGGTAGTGTGTATCCTG) Kun cancer_hyb_sept14 51,639 110-130bp V8(T*C*TAATCTAGCGCGACGTCU//5Phos/CCACAAGAGGCGCTATG) Kun LMS_selector 17,342 124-130bp NE(TGCCTAGGACCGGATCAACT/GCTTCGGTTCACGCAATG) Kun padlock_SNPs 12,974 125bp V4(G*A*CTGGAAGAGCACTGTU//5Phos/AGCCTCATGCGTATCCG) **has mismatch with Noi's V4?? Total 90,000 {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Oligo calculation for ePCR''' | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''''' |- | gram-conc. (Tube concentration)||48.43||ng/ul|| |- | Tube volume||80||ul|| |- | Total amount||3874.4||ng|| |- | Oligo average length||125||nt||* The average size for all probes in this pool |- | MW (Length*330 Da/bp)||41250||Da (g/mole)|| |- | Mole-conc. (gram-conc / MW * 1000)||1.174060606||uM|| |- | |||||| |- | |} Set frac. Cor.nM Amp. Primers quick-Run(Ct up) Oligo set 1 0.09 104.95 V6 15 Oligo set 2 0.57 673.64 V8 7 Oligo set 3 0.19 226.23 NE 11 Oligo set 4 0.14 169.25 V4 14 ===Expansion PCR=== ====Quick run with 1ul for each primer set==== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Tube ID''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''[nM]''' | align="center" style="background:#f0f0f0;"|'''[ul]''' | align="center" style="background:#f0f0f0;"|'''AmpP''' | align="center" style="background:#f0f0f0;"|'''Ct up''' | align="center" style="background:#f0f0f0;"|'''Ct stop''' | align="center" style="background:#f0f0f0;"|'''final Flu.''' |- | s1||T21.v4 ePCR||361||0.5||v4 **||1||6||0.32 |- | s2||oligo mix||~ 169||1||v4 **||13||20||~0.2 |- | s3||T21.v6 ePCR||181||1||v6||1||7||0.28 |- | s4||oligo mix||~105||1||v6||15||20||~0.13 |- | s5||selector ePCR||150||1||eMIP||no||20||no |- | s6||oligo mix||~226||1||eMIP||no||20||no |} [[File:ePCR_QPCR.jpg|400px]][[File:ePCR_PAGE.jpg|400px]] ====Recheck with eMIP primer sets==== * Positive control used is the pPCR product (65nM is confirmed by Nanodrop again) of selector set last time * Probe/primer were also confirmed with PAGE together with PCR product * The problem may be due to eMIP primer set -> re-order [[File:quickRun_eMIP_QPCR.jpg|400px]][[File:quickRun_eMIP_PAGE.jpg|400px]] * Fortunately, I found another eMIP pair from my box, so I gave this set a try with PC used above. * It works as expected, so I toss the bad primers originally in Noi's box. [[File:ePCR_eMIP_QPCR.jpg|400px]][[File:ePCR_v4.jpg|400px]][[File:quickRun_PAGE.jpg|400px]][[File:ePCR_eMIP.jpg|400px]] ===Production PCR for 3 oligo sets 2-4 (v4,v8,eMIP)=== [[File:pPCR_v4_QPCR.jpg|400px]][[File:pPCR_v3_QPCR.jpg|400px]]
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