Matt:LabNotes/2015-4-28

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Revision as of 20:27, 28 April 2015 by >Mzcai (Created page with "==in vitro Capture with CA12k_Nov2014 Probe Set + Supp Oligos== ===HBRR cDNA Synthesis=== * Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-...")
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in vitro Capture with CA12k_Nov2014 Probe Set + Supp Oligos

HBRR cDNA Synthesis

  • Followed NEB E6300S protocol: [1]
  • Human Brain Reference RNA: [2]
    • Labeled concentration = 1ug/ul
  • 2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control
Components Volume
RNA 0.5 ul (0.5 ug)
d(T)23VN (50 µM) 2 ul
H2O 5.5 ul
Total 8 ul
  • Denatured RNA for 5 min at 70C and then put on ice
  • Added the following to tubes
Components Volume
M-MuLV Reaction Mix 10 ul
M-MuLV Enzyme Mix 2 ul
Total 20 ul
  • Incubated for one hour at 42C
  • Inactivated enzyme for 5 min at 80C
  • Purified cDNA in one Qiagen PCR clean up column and NTC in another
    • Eluted 30ul each

Calculate Probes Needed

V4

Probe:target 1000:1 '
Probe size 3514 probes
DNA templet 300 ng
gDNA MW 1.95x10^12 g/mol
gDNA (300ng) 1.5385x10^-19 mol
Probe (1000:1) 1.5385x10^-16 mol
Probe MW (3514, 150nt) 1.71585106x10^8 g/mol
Amount Probe req'd 26.4 ng

V7

Probe:target 1000:1 '
Probe size 2486 probes
DNA templet 300 ng
gDNA MW 1.95x10^12 g/mol
gDNA (300ng) 1.5385x10^-19 mol
Probe (1000:1) 1.5385x10^-16 mol
Probe MW (2486, 150nt) 1.21388894x10^8 g/mol
Amount Probe req'd 18.7 ng