Matt:LabNotes/2015-5-5: Difference between revisions
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>Mzcai m (→Production PCR) |
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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | ||
[[File:022815_CA12kNov14_ProductionPCR_V4.JPG | 800px]] | |||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
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**80ul 3M NaOAc pH 5.2-5.5 | **80ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for 30min | *Vortexed and put in -80C for 30min<!-- | ||
*Centrifuged at 3000rpm at 4C for 30 min | *Centrifuged at 3000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH |
Revision as of 21:42, 5 May 2015
CA12k_Nov2014_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon CA12k_Nov14_V4 (10nM) | 0.2 | 20 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (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:022815 CA12kNov14 ProductionPCR V4.JPG
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each) for V4
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30min