Matt:LabNotes/2015-5-5: Difference between revisions

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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]]
[[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 PCR

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