Matt:LabNotes/2014-5-22

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Revision as of 01:04, 23 May 2014 by >Mzcai (→‎Probe Expansion PCR)
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Agi26k0gap Probe Production

  • Use 20nM aliquot of Agilent oligos in tube labeled "FISHYB V1 probes 5/07/13"

Probe Expansion PCR

  • Expansion PCR in 200ul total: 2 tubes of 100ul for 0bp gap probes with AP1V6 primers
' 0 gap probes (ul)
Agi26k (20nM) 10
2x Kapa SYBG qPCR MM 100
100uM pAP1V6U 0.8
100uM AP2V6 0.8
H2O 88.4
Total 200
  • Mixed thoroughly and then split into two tubes

94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 14 -> 72C 3min -> 15C hold

  • Stopped after 12 cycles

File:20140522 Agi26k0gapExpansionPCR.JPG

  • Purified with 2 Qiagen columns
  • Eluted with 40ul each tube and combined into two tubes (80ul total)
  • Nanodrop: 32.2 ng/ul -> 32.2ng/ul / (228bp*660Da/bp) = 214nM
  • Dilute 2.34ul in 47.66ul H2O
  • Final volumes:
    • 75ul of 214nM
    • 50ul of 10nM

Production PCR