Matt:LabNotes/2015-4-28
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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 |