Matt:LabNotes/2017-4-12: Difference between revisions

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>Mzcai
(Created page with "==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8 ===Production PCR=== *[[Matt:LabNotes/2017-3-30 | 25nM 1st round amplicon...")
 
>Mzcai
Line 10: Line 10:
| align="center" style="background:#f0f0f0;"|'''Volume (100X)'''
| align="center" style="background:#f0f0f0;"|'''Volume (100X)'''
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| First round amplicon CA12k_Nov14_V4 (10nM)||0.1||10
| First round amplicon Agi15k_Feb2017_V4 (25nM)||0.1||10
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| 2X KAPA SYBG MM||50||5000
| 2X KAPA SYBG MM||50||5000
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| AP2V4 (100uM)||0.4||40
| AP2V4 (100uM)||0.4||40
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| H2O||49||4900
| H2O||49||4910
|-
|-
| Total||100||10000
| Total||100||10000

Revision as of 21:16, 12 April 2017

Agi15k_Feb2017_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon Agi15k_Feb2017_V4 (25nM) 0.1 10
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4910
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[ | 800px]]

EtOH Precipitation

  • 8 5-ml tubes (with 12 wells of PCR product each) for V4
    • 1200ul PCR product
    • 3000ul 100% EtOH
    • 3ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight