Matt:LabNotes/2014-5-22
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Agi26k0gap Probe Production
- Reference: Matt:LabNotes/2013-5-10
- Use 20nM aliquot of Agilent oligos in tube labeled "FISHYB V1 probes 5/07/13"
Probe Expansion PCR
- Expansion PCR in 200ul total: 2 tubes of 100ul for 0bp gap probes with AP1V6 primers
' | 0 gap probes (ul) |
Agi26k (20nM) | 10 |
2x Kapa SYBG qPCR MM | 100 |
100uM pAP1V6U | 0.8 |
100uM AP2V6 | 0.8 |
H2O | 88.4 |
Total | 200 |
- Mixed thoroughly and then split into two tubes
94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 14 -> 72C 3min -> 15C hold
- Stopped after 12 cycles
- Purified with 2 Qiagen columns
- Eluted with 40ul each tube and combined into two tubes (80ul total)
- Nanodrop: __ ng/ul -> __ng/ul / (228bp*660Da/bp) = __nM
- Final Volume of __ul for each first round amplicon