Rui:LabNotes/Haplotyping/2014-11-12
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Oligo set 90k_oligos_30Sept2014[edit]
- 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[edit]
*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
Oligo calculation for ePCR | Content | Unit | ' |
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[edit]
Quick run with 1ul for each primer set[edit]
Tube ID | Sample | [nM] | [ul] | AmpP | Ct up | Ct stop | 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.jpgFile:EPCR PAGE.jpg
Recheck with eMIP primer sets[edit]
- 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.jpgFile:QuickRun eMIP PAGE.jpg
- 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.jpgFile:EPCR v4.jpgFile:QuickRun PAGE.jpgFile:EPCR eMIP.jpg