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

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*Wash the pellet with 500ul Buffer QX1
*Wash the pellet with 500ul Buffer QX1
*Resuspend the pellet by vortexing
*Resuspend the pellet by vortexing
*Transfer to 1.5ml tubes
*Centrifuge sample for 30sec and remove supernatant again with pipette
*Centrifuge sample for 30sec and remove supernatant again with pipette
*Wash the pellet twice with 500ul Buffer PE
*Wash the pellet twice with 500ul Buffer PE

Revision as of 01:47, 4 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
  • Transfer to 1.5ml tubes
  • 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: