Matt:LabNotes/2015-2-5: Difference between revisions

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>Mzcai
(Created page with "==CA12k_Nov2014_V7 Probe Production== *Production of Padlock Probes (V7) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering *Do double of what I normally do (96 ...")
 
>Mzcai
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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 overnight<!--
*Vortexed and put in -80C for 90min
*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
Line 40: Line 40:
*Discarded supernatant and air-dried for 5 min in hood
*Discarded supernatant and air-dried for 5 min in hood
*Resuspended DNA with 100ul H2O
*Resuspended DNA with 100ul H2O
 
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===Qia Column Purification===
===Qia Column Purification===
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol

Revision as of 02:30, 6 February 2015

CA12k_Nov2014_V7 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V7U (100uM) 0.4 40
AP2V7 (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: | 800px]]

EtOH Precipitation

  • 12 5-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 90min
  • Centrifuged at 3000rpm at 4C for 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O