Matt:LabNotes/2015-6-3: Difference between revisions

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>Mzcai
(Created page with "==CA12k_Nov2014_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering ===Production PCR=== *[[Matt:LabNote...")
 
>Mzcai
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===QIAEX-II Purification===
===QIAEX-II Purification===
*Divide into 12 5mL tubes of 800ul each
*Divide into 8 5mL tubes of 1200ul each
*Add 2400ul (3X volume) of Buffer QX1
*Add 3600ul (3X volume) of Buffer QX1
*Vortex the QIAEXII for 30sec
*Vortex the QIAEXII for 30sec
*Add 30ul QIAEXII (30ul for every 10ug)
*Add 30ul QIAEXII (30ul for every 10ug)
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*Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
*Repeat 20ul H2O resuspension and incubation to improve yield 10-15%


*Total volume: 480ul
*Total volume: 320ul
*Nanodrop quantification:
*Nanodrop quantification:
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Revision as of 23:26, 3 June 2015

CA12k_Nov2014_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_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 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold

QIAEX-II Purification

  • Divide into 8 5mL tubes of 1200ul each
  • Add 3600ul (3X volume) of Buffer QX1
  • Vortex the QIAEXII for 30sec
  • Add 30ul QIAEXII (30ul for every 10ug)
  • Incubate at 50C while mixing for 10min
    • Color should be yellow
  • Centrifuge sample for 30sec
  • Remove supernatant with a pipette
  • Wash the pellet with 500ul Buffer QX1
  • Resuspend the pellet by vortexing
  • Centrifuge sample for 30sec and remove supernatant again with pipette
  • Wash the pellet twice with 500ul Buffer PE
  • Air-dry the pellet for 30min until the pellet turns white
  • To elute, resuspend with 20ul H2O and vortexing
  • Incubate at RT for 5min
  • Centrifuge for 30sec and pipette out supernatant with sample
  • Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
  • Total volume: 320ul
  • Nanodrop quantification: