Matt:LabNotes/2014-12-18
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CA12k_Nov2014 Probe Preparation[edit]
- Probe Design
- Received 190-mer oligos 12/18/14
- Suspended in 80ul TE Buffer
- Name: Fisga_Nov2014_seq
- Conc: 52.86 ng/ul (4.23 ug total)
- Calculate MW of 190 nt probe = 190nt * 308.9D/nt + 79D = 58,770 g/mol
- 52.86 ng/ul * pmol/58.770 ng = 0.8994 pmol/ul = ~0.9 uM
Aliquot[edit]
- Aliquot 40ul into 1.5ml tube
- From the aliquot take 1ul and dilute with H2O to 22.5ul in 1.5ml tube (40nM final conc)
900nM x 1ul = 40nM x 22.5ul
Expansion PCR Test[edit]
- NTC
Component | Volume |
Seed oligo | 0 |
F/R V6 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 10.5 |
Total | 25 |
- +ve Control
Component | Volume |
V6 +ve (2nM) | 10.5 |
F/R V6 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 0 |
Total | 25 |
- 1nM
Component | Volume |
Seed oligo (40nM) | 0.6 |
F/R V6 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 9.9 |
Total | 25 |
- 10nM
Component | Volume |
Seed oligo (40nM) | 6.25 |
F/R V6 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 4.25 |
Total | 25 |
- 100nM
Component | Volume |
Seed oligo (900nM) | 2.8 |
F/R V6 Primer Mix (10uM) | 2 |
2X Kapa MM | 12.5 |
H2O | 7.7 |
Total | 25 |
Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 22 -> 72C 2min -> 15C hold
File:121814 CA12kNov2014 ExpansionPCRTest.JPG
- Noi recommended doing 21 cycles at 100nM for Expansion PCR