Matt:LabNotes/2017-4-12: Difference between revisions
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>Mzcai (Created page with "==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8 ===Production PCR=== *[[Matt:LabNotes/2017-3-30 | 25nM 1st round amplicon...") |
>Mzcai m (→Production PCR) |
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| align="center" style="background:#f0f0f0;"|'''Volume (100X)''' | | align="center" style="background:#f0f0f0;"|'''Volume (100X)''' | ||
|- | |- | ||
| First round amplicon | | First round amplicon Agi15k_Feb2017_V4 (25nM)||0.1||10 | ||
|- | |- | ||
| 2X KAPA SYBG MM||50||5000 | | 2X KAPA SYBG MM||50||5000 | ||
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| AP2V4 (100uM)||0.4||40 | | AP2V4 (100uM)||0.4||40 | ||
|- | |- | ||
| H2O||49|| | | H2O||49||4910 | ||
|- | |- | ||
| Total||100||10000 | | Total||100||10000 |
Revision as of 21:16, 12 April 2017
Agi15k_Feb2017_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8
Production PCR
- 25nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon Agi15k_Feb2017_V4 (25nM) | 0.1 | 10 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4910 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[ | 800px]]
EtOH Precipitation
- 8 5-ml tubes (with 12 wells of PCR product each) for V4
- 1200ul PCR product
- 3000ul 100% EtOH
- 3ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight