Noi/NOTES/2013-2-27: Difference between revisions

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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
== Plan for probe preparation for Matt's project ==
== Plan for probe preparation for Matt's project ==
* I will be preparing probes using the same method as I did for LC Sciences oligoes, [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-10-6| LC_Sciences probe prep]]
* I will be preparing probes using the same method as I did for LC Sciences oligoes, [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-10-6]]
* Check reagents to make sure I have all reagents ready for probe prep and padlock capture
* Check reagents to make sure I have all reagents ready for probe prep and padlock capture
  '''Probe prep'''
  '''Probe prep'''
  - Waiting for seed oligo: expected to come next Wednesday (March6, 2013)
  - Waiting for seed oligos: expected to come next Wednesday (March6, 2013)
  - eMIP_CA1_F and eMIP_CA1_R primer for expansion PCR and production PCR (very little left, ordered 2/28/2013)
  - eMIP_CA1_F and eMIP_CA1_R primer for expansion PCR and production PCR (very little left, ordered 2/28/2013)  
  ; arrived 3/04/2013 -> resuspend 100uM
  - 2x KAPA SYBG FAST qPCR MM
  - 2x KAPA SYBG FAST qPCR MM
  - Nt.AlwI (NEB) and Nb.BsrDI (NEB) -> ordered 2/26/2013
  - Nt.AlwI (NEB) and Nb.BsrDI (NEB) -> ordered 2/26/2013 ; arrived 3/04/2013
  - Qiaquick colums, TBU gel, Nanosep column
  - Qiaquick columns, TBU gel, Nanosep columns
  '''Padlock capture'''
  '''Padlock capture'''
  Note: There is no gap for this capture, so only need Ampligase
  Note: There is no gap for this capture, so only need Ampligase
  - Ampligase and buffer
  - Ampligase and buffer: yes
  - ExoI/ExoIII
  - ExoI/ExoIII: yes
  - Amplification primers (Matt)
  - Amplification primers (Matt)
* Look at the probe design (primer region), it's pretty much the same as the probes synthesized by LC Sciences oligoes. Start at '''AGGACCGGATCAACT'''
* Need to check with Dr. Zhang about the gDNA, RNA and cDNA amount in capture. The expected size of citcularized DNA (no gap --> 170bp plus length of Illumina adapter = XX bp? look at primer design)
* Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-6

Latest revision as of 22:50, 6 March 2013

Plan for probe preparation for Matt's project[edit]

  • I will be preparing probes using the same method as I did for LC Sciences oligoes, [[1]]
  • Check reagents to make sure I have all reagents ready for probe prep and padlock capture
Probe prep
- Waiting for seed oligos: expected to come next Wednesday (March6, 2013)
- eMIP_CA1_F and eMIP_CA1_R primer for expansion PCR and production PCR (very little left, ordered 2/28/2013) 
  ; arrived 3/04/2013 -> resuspend 100uM
- 2x KAPA SYBG FAST qPCR MM
- Nt.AlwI (NEB) and Nb.BsrDI (NEB) -> ordered 2/26/2013 ; arrived 3/04/2013
- Qiaquick columns, TBU gel, Nanosep columns
Padlock capture
Note: There is no gap for this capture, so only need Ampligase
- Ampligase and buffer: yes
- ExoI/ExoIII: yes
- Amplification primers (Matt)
  • Look at the probe design (primer region), it's pretty much the same as the probes synthesized by LC Sciences oligoes. Start at AGGACCGGATCAACT
  • Need to check with Dr. Zhang about the gDNA, RNA and cDNA amount in capture. The expected size of citcularized DNA (no gap --> 170bp plus length of Illumina adapter = XX bp? look at primer design)
  • Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-6