Matt:LabNotes/2017-3-30
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Agilent 15k Feb2017 Oligos[edit]
- Probe design
- Oligonucleotide Library received (Part Number G7221A)
- Dan Konet says the pools yield ~10pmols
- I resuspended to 200ul with nf-H2O (50nM concentration if actually 10pmols)
Probe Expansion PCR[edit]
- Expansion PCR in 4 tubes of 100ul
- V4 primers for Human Brain and V7 primers for Mouse Brain
' | 0 gap probes (ul) | 20 gap probes (ul) |
Agi15k (50nM) | 10 | 10 |
2x Kapa SYBG qPCR MM | 100 | 100 |
100uM pAP1V7U | 0.8 | 0 |
100uM AP2V7 | 0.8 | 0 |
100uM pAP1V4U | 0 | 0.8 |
100uM AP2V4 | 0 | 0.8 |
H2O | 88.4 | 88.4 |
Total | 200 | 200 |
94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 14 -> 72C 3min -> 15C hold
- Stopped after 11 cycles
- V7 could probably be stopped 2 cycles earlier but couldn't tell because qPCR software did weird "baseline subtraction" that messed with curve
- Figured out to turn off baseline subtraction in settings for next time
File:033017 ExpansionPCR Agi15k.jpg
- Purified with 2 Qiagen columns for each of the two probe sets
- Eluted with 50ul each tube and combined into two tubes (100ul for each of the two probe sets)
- Nanodrop:
- V4: 42.2 ng/ul -> 42.2ng/ul / (195bp*660Da/bp) = 328nM
- V7: 48.2 ng/ul -> 48.2ng/ul / (195bp*660Da/bp) = 375nM
- Dilute with H2O to 25nM
- V4: Add 1,188 H2O
- V7: Add 1,372 H2O