Matt:LabNotes/2015-4-28: Difference between revisions
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*Sample 4, accidentally added an extra 9ul H2O so added 4ul 10X Ampligase Buffer | *Sample 4, accidentally added an extra 9ul H2O so added 4ul 10X Ampligase Buffer | ||
**Added 4ul of AmpLigase enzyme mix as well (vs 3ul for others) | |||
*Add 40ul mineral oil on top | *Add 40ul mineral oil on top | ||
Revision as of 19:19, 30 April 2015
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
- Nanodrop w/ dsDNA setting:
- cDNA: 30.7 ng/ul
- Neg Ctrl: 10.2 ng/ul
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 |
Probes, Target and Ampligase Buffer Mix
- gDNA: 12878 (80.3ng/ul)
- First strand cDNA from HBRR
- CA12k_Nov2014 Probes
- V4 819nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 40.0 ng/ul
- V7 768nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 37.5 ng/ul
- Diluted 1:2 with H2O for easier pipetting (final conc 20.0ng/ul and 18.75ng/ul)
- NTC is the NTC from first strand cDNA synthesis
- 5nM of each supp oligos (5 x 10^-6 nmol/ul)
- 100-fold more of each suppressor oligo = 1.5385 x 10^-14 mol = 15 x 10^-6 nmol
Sample # | Sample Description | Probes | Target | 5nM Supp Oligos | 10X Ampligase Buffer | H2O | Total |
1 | V4 - gDNA | 1.3 | 3.8 | 3 | 3 | 18.9 | 30 |
2 | V4 - cDNA | 1.3 | 15 | 3 | 3 | 7.7 | 30 |
3 | V4 - NTC | 1.3 | 15 | 3 | 3 | 7.7 | 30 |
4 | V7 - gDNA | 1 | 3.8 | 3 | 3 | 19.2 | 30 |
5 | V7 - cDNA | 1 | 15 | 3 | 3 | 8 | 30 |
6 | V7 - NTC | 1 | 15 | 3 | 3 | 8 | 30 |
- Sample 4, accidentally added an extra 9ul H2O so added 4ul 10X Ampligase Buffer
- Added 4ul of AmpLigase enzyme mix as well (vs 3ul for others)
- Add 40ul mineral oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
AmpLigase enzyme mix
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 30ul |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 2.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 2.00 |
H2O | 16.00 | ||||
Total | 20.00 |