Noi/NOTES/2013-2-27
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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