Matt:LabNotes/2014-12-18

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CA12k_Nov2014 Probe Preparation[edit]

  • Probe Design
  • Received 190-mer oligos 12/18/14
    • Suspended in 80ul TE Buffer
    • Name: Fisga_Nov2014_seq
    • Conc: 52.86 ng/ul (4.23 ug total)
      • Calculate MW of 190 nt probe = 190nt * 308.9D/nt + 79D = 58,770 g/mol
    • 52.86 ng/ul * pmol/58.770 ng = 0.8994 pmol/ul = ~0.9 uM

Aliquot[edit]

  • Aliquot 40ul into 1.5ml tube
  • From the aliquot take 1ul and dilute with H2O to 22.5ul in 1.5ml tube (40nM final conc)
 900nM x 1ul = 40nM x 22.5ul

Expansion PCR Test[edit]

  • NTC
Component Volume
Seed oligo 0
F/R V6 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 10.5
Total 25
  • +ve Control
Component Volume
V6 +ve (2nM) 10.5
F/R V6 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 0
Total 25
  • 1nM
Component Volume
Seed oligo (40nM) 0.6
F/R V6 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 9.9
Total 25
  • 10nM
Component Volume
Seed oligo (40nM) 6.25
F/R V6 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 4.25
Total 25
  • 100nM
Component Volume
Seed oligo (900nM) 2.8
F/R V6 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 7.7
Total 25

Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 22 -> 72C 2min -> 15C hold

File:121814 CA12kNov2014 ExpansionPCRTest.JPG

  • Noi recommended doing 21 cycles at 100nM for Expansion PCR