Matt:LabNotes/2017-5-20: Difference between revisions

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(Created page with "==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8 ===Notes from last time=== *Do 12 or 13 cycle...")
 
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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold<br>
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold<br>
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[[File:20170412_Agi15kFeb2017_ProductionPCR.png | 600px]]
*Do 12 or 13 cycles next time


===EtOH Precipitation===
===EtOH Precipitation===
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**120ul 3M NaOAc pH 5.2-5.5
**120ul 3M NaOAc pH 5.2-5.5


*Vortexed and put in -80C for overnight
*Vortexed and put in -80C for overnight<!--
*Centrifuged at 10,000rpm at 4C for 30 min
*Centrifuged at 10,000rpm 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 23:08, 20 May 2017

Agi15k_Feb2017_V4 Probe Production

Notes from last time

  • Do 12 or 13 cycles Production PCR
  • Experiment with 1hr vs 2hr Lambda digestion
  • Think of way to maximize USER and DpnII digestion

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 13 -> 72C 2min -> 15C hold

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