Matt:LabNotes/2015-10-19
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CA12k_Nov2014 Expansion PCR[edit]
- First time doing expansion PCR on this oligo pool
- Only 15ul left so using the rest
Expansion PCR[edit]
- Matt:LabNotes/2014-12-18 conclusion: 21 cycles of PCR starting with 100nM seed oligos
- Divide into 2 Biorad low qPCR tubes with 67.5ul each
- V4 primers - CA12k_Nov2014_V4
' | Contig of Exons (V4) |
CA12k_Nov2014 Seed Oligos (900nM) | 15 |
2X KAPA SYBG MM | 67.5 |
100uM AP1V4U | 0.6 |
100uM AP2V4 | 0.6 |
H2O | 51.3 |
Total | 135 |
Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold
File:20151019 ExpansionPCR CA12kNov14V4.PNG
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V4: 5.3ng/ul -> 5.3ng/ul / (188bp*660Da/bp) = 42.7nM
- Diluted to 10nM
- V4: 100ul x 42.7nM = 427ul x 10nM
- Add 327ul H2O
- V4: 100ul x 42.7nM = 427ul x 10nM
Update Jan 26 2016[edit]
- Remeasured concentration of seed oligos due to low yields after 15 cycles of Production PCR
- Erin took 10ul for Qubit dsDNA measurement
- 168 ng/mL = ~1nM