Matt:LabNotes/2013-5-10

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Revision as of 21:07, 11 May 2013 by >Mzcai (→‎Probe Expansion PCR)
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Agilent Probe Prep[edit]

  • Dr. Zhang resuspended probes to 20nM
  • Dr. Zhang aliquoted Agilent probes into separate tube labelled "FISHYB V1 probes 5/07/13"

Probe Expansion PCR[edit]

Expansion PCR in 2 tubes of 180ul total (1 tube for 0bp gap probes with AP1V6 primers; 1 tube for 20bp gap probes with AP1V4 primers)

' 0 gap probes (ul) 20 gap probes (ul)
Agi26k (20nM) 10 10
2x Kapa SYBG qPCR MM 100 100
100uM pAP1V6U 0.8 0
100uM AP2V6 0.8 0
100uM pAP1V4U 0 0.8
100uM AP2V4 0 0.8
H2O 88.4 88.4
Total 200 200
  • NOTE: Mixed thoroughly and then removed 20ul so there would be room to close lid

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

  • Stopped after 12 cycles

File:051013 ExpansionPCR Agi26k.jpg

  • Purified with 2 Qiagen columns for each of the two probe sets
  • Eluted with 40ul each tube and combined into two tubes (80ul for each of the two probe sets)
  • Nanodrop:
    • 0gap: 16.90 ng/ul -> 16.9ng/ul / (228bp*660Da/bp) = 112nM
    • 20gap: 18.46 ng/ul -> 18.46ng/ul / (228bp*660Da/bp) = 123nM
  • Final Volume of 48.5ul for each first round amplicon

Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-11