Matt:LabNotes/2015-4-28: Difference between revisions

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**V4 819nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 40.0 ng/ul
**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
**V7 768nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 37.5 ng/ul
*50nM of each supp oligos (50 x 10^-6 nmol/ul)
***Diluted 1:2 with H2O for easier pipetting (final conc 20.0ng/ul and 18.75ng/ul)
***1000-fold more of each suppressor oligo = 1.5385 x 10^-13 mol = 1.5 x 10^-4 nmol
*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


{| {{table}}
{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Probes'''
| align="center" style="background:#f0f0f0;"|'''Probes'''
| align="center" style="background:#f0f0f0;"|'''Target'''
| align="center" style="background:#f0f0f0;"|'''Target'''
| align="center" style="background:#f0f0f0;"|'''5nM Supp Oligos'''
| align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer'''
| align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer'''
| align="center" style="background:#f0f0f0;"|'''H2O'''
| align="center" style="background:#f0f0f0;"|'''H2O'''
| align="center" style="background:#f0f0f0;"|'''Total'''
| align="center" style="background:#f0f0f0;"|'''Total'''
|-
|-
| 1||V4 - gDNA||2.5||3.8||3||20.7||30
| 1||V4 - gDNA||1.3||3.8||3||3||18.9||30
|-
|-
| 2||V4 - cDNA||2.5||3.8||3||20.7||30
| 2||V4 - cDNA||1.3||15||3||3||7.7||30
|-
|-
| 3||V4 - NTC||2.5||0||3||24.5||30
| 3||V4 - NTC||1.3||15||3||3||7.7||30
|-
|-
| 4||V7 - gDNA||1||15||3||11||30
| 4||V7 - gDNA||1||3.8||3||3||19.2||30
|-
|-
| 5||V7 - cDNA||1||15||3||11||30
| 5||V7 - cDNA||1||15||3||3||8||30
|-
|-
| 6||V7 - NTC||1||0||3||26||30
| 6||V7 - NTC||1||15||3||3||8||30
|}
|}



Revision as of 20:08, 29 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

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
  • Add 30ul mineral oil on top