Matt:LabNotes/2016-7-26

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Revision as of 18:39, 26 July 2016 by >Mzcai (Created page with "=TB12k_Apr2016 V4 Probe Preparation= * 10nM Seed Oligos of TB12k_Apr2016 V4 ===Production PCR=== *Make 10ml V4 master mix for 96 wells {| {{table...")
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TB12k_Apr2016 V4 Probe Preparation

Production PCR

  • Make 10ml V4 master mix for 96 wells
Components Volume (1X) Volume (100X)
First round amplicon TB12k_Apr2016_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 10,000

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold

  • Did in regular thermocyclers

EtOH Precipitation

  • 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5