Matt:LabNotes/2013-5-10
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Agilent Probe Prep[edit]
- Dr. Zhang resuspended probes to 20nM
- Dr. Zhang aliquoted Agilent probes into separate tube labelled "FISHYB V1 probes 5/07/13"
Probe Expansion PCR[edit]
Expansion PCR in 2 tubes of 180ul total (1 tube for 0bp gap probes with AP1V6 primers; 1 tube for 20bp gap probes with AP1V4 primers)
' | 0 gap probes (ul) | 20 gap probes (ul) |
Agi26k (20nM) | 10 | 10 |
2x Kapa SYBG qPCR MM | 100 | 100 |
100uM pAP1V6U | 0.8 | 0 |
100uM AP2V6 | 0.8 | 0 |
100uM pAP1V4U | 0 | 0.8 |
100uM AP2V4 | 0 | 0.8 |
H2O | 88.4 | 88.4 |
Total | 200 | 200 |
- NOTE: Mixed thoroughly and then removed 20ul so there would be room to close lid
94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 14 -> 72C 3min -> 15C hold
- Stopped after 12 cycles
File:051013 ExpansionPCR Agi26k.jpg
- Purified with 2 Qiagen columns for each of the two probe sets
- Eluted with 40ul each tube and combined into two tubes (80ul for each of the two probe sets)
- Nanodrop:
- 0gap: 16.90 ng/ul -> 16.9ng/ul / (228bp*660Da/bp) = 112nM
- 20gap: 18.46 ng/ul -> 18.46ng/ul / (228bp*660Da/bp) = 123nM
- Final Volume of 48.5ul for each first round amplicon
Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-11