Matt:LabNotes/2017-3-30

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Agilent 15k Feb2017 Oligos[edit]

  • Probe design
  • Oligonucleotide Library received (Part Number G7221A)
    • Dan Konet says the pools yield ~10pmols
  • I resuspended to 200ul with nf-H2O (50nM concentration if actually 10pmols)

Probe Expansion PCR[edit]

  • Expansion PCR in 4 tubes of 100ul
    • V4 primers for Human Brain and V7 primers for Mouse Brain
' 0 gap probes (ul) 20 gap probes (ul)
Agi15k (50nM) 10 10
2x Kapa SYBG qPCR MM 100 100
100uM pAP1V7U 0.8 0
100uM AP2V7 0.8 0
100uM pAP1V4U 0 0.8
100uM AP2V4 0 0.8
H2O 88.4 88.4
Total 200 200

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

  • Stopped after 11 cycles
    • V7 could probably be stopped 2 cycles earlier but couldn't tell because qPCR software did weird "baseline subtraction" that messed with curve
    • Figured out to turn off baseline subtraction in settings for next time

File:033017 ExpansionPCR Agi15k.jpg

  • Purified with 2 Qiagen columns for each of the two probe sets
  • Eluted with 50ul each tube and combined into two tubes (100ul for each of the two probe sets)
  • Nanodrop:
    • V4: 42.2 ng/ul -> 42.2ng/ul / (195bp*660Da/bp) = 328nM
    • V7: 48.2 ng/ul -> 48.2ng/ul / (195bp*660Da/bp) = 375nM
  • Dilute with H2O to 25nM
    • V4: Add 1,188 H2O
    • V7: Add 1,372 H2O