Matt:LabNotes/2015-1-13: Difference between revisions

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(Created page with "==CA12k_Nov2014_V7 Probe Production== *Production of Padlock Probes targeting constitutive exon cDNA (V7 primers) of [[Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_orde...")
 
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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
*Stopped after 15 cycles
[[File:011214_CA12kNov14_ProductionPCR_V7.JPG | 800px]]
[[File: | 800px]]


===EtOH Precipitation===
===EtOH Precipitation===
*6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
*6 15-ml tubes (with 8 wells of PCR product each) for V7
**800ul PCR product
**800ul PCR product
**2000ul 100% EtOH
**2000ul 100% EtOH

Revision as of 04:21, 13 January 2015

CA12k_Nov2014_V7 Probe Production

Production PCR

Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V7U (100uM) 0.4 20
AP2V7 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:011214 CA12kNov14 ProductionPCR V7.JPG

EtOH Precipitation

  • 6 15-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