Matt:LabNotes/2015-1-12: Difference between revisions

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(Created page with "==Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 Probe Set== * Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis...")
 
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* Universal Human Reference RNA: [[Matt:LabNotes/2014-9-25#Purify_UHRR_of_DNA | Previously purified of contaminating DNA]]
* Universal Human Reference RNA: [[Matt:LabNotes/2014-9-25#Purify_UHRR_of_DNA | Previously purified of contaminating DNA]]
** About 3ul remaining from previous experiments
** About 2ul remaining from previous experiments
** Estimate concentration ~0.5ug/ul
** Estimate concentration ~0.5ug/ul


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| align="center" style="background:#f0f0f0;"|'''  Volume  '''
| align="center" style="background:#f0f0f0;"|'''  Volume  '''
|-
|-
| RNA||1.5 ul (0.75 ug)
| RNA||1 ul (0.5 ug)
|-
|-
| d(T)23VN (50 µM)||2 ul
| d(T)23VN (50 µM)||2 ul
|-
|-
| H2O||4.5 ul
| H2O||5 ul
|-
|-
| Total||8 ul
| Total||8 ul
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* Incubated for one hour at 42C
* Incubated for one hour at 42C
* Inactivated enzyme for 5 min at 80C
* Inactivated enzyme for 5 min at 80C
*Purified cDNA in one Qiagen PCR clean up column and NTC in another
**Eluted 30ul each and stored in -20C

Latest revision as of 02:19, 13 January 2015

Make First Strand cDNA for in vitro Capture with CA12k_Nov2014 Probe Set[edit]

  • Followed NEB E6300S protocol: [1]
  • 2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control
Components Volume
RNA 1 ul (0.5 ug)
d(T)23VN (50 µM) 2 ul
H2O 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 and stored in -20C