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

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(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-...")
 
>Mzcai
mNo edit summary
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**Eluted 30ul each
**Eluted 30ul each


*Nanodrop:
**cDNA:
**Neg Ctrl:
===Calculate Probes Needed===
===Calculate Probes Needed===
====V4====
====V4====
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| Amount Probe req'd||18.7||ng
| Amount Probe req'd||18.7||ng
|}
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===Probes, Target and Ampligase Buffer Mix===
===Probes, Target and Ampligase Buffer Mix===
*gDNA: 12878 (80.3ng/ul)
*gDNA: 12878 (80.3ng/ul)
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| 6||V7 - NTC||1||0||3||26||30
| 6||V7 - NTC||1||0||3||26||30
|}
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'''Program'''<br>
'''Program'''<br>
* 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
* 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h

Revision as of 21:59, 28 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:
    • cDNA:
    • Neg Ctrl:

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

Sample # Sample Description Probes Target 10X Ampligase Buffer H2O Total
1 V4 - gDNA 2.5 3.8 3 20.7 30
2 V4 - cDNA 2.5 3.8 3 20.7 30
3 V4 - NTC 2.5 0 3 24.5 30
4 V7 - gDNA 1 15 3 11 30
5 V7 - cDNA 1 15 3 11 30
6 V7 - NTC 1 0 3 26 30