Matt:LabNotes/2015-2-5
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CA12k_Nov2014_V7 Probe Production
- Production of Padlock Probes (V7) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
- Do double of what I normally do (96 100ul PCR reactions instead of 48)
Production PCR
- 10nM 1st round amplicons
- V7 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V7 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V7U (100uM) | 0.4 | 40 |
AP2V7 (100uM) | 0.4 | 40 |
H2O | 49 | 4900 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each) for V7
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight