Sam:LabNotes/Microbiome-new/2010-5-25
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Pobes design for specific capturing on unculturable mouse gut bacteria[edit]
Background[edit]
- First mouse gut single cell genome was sugeneced and de novo assembled.
- Sample information: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-1-28
- Solexa library information: http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-3-2
- The assembled genome contigs that are larger than 1kb were used for probes designing.
- I pick up three largest contings as template. Two probes were designed on each contig.
- The universal FISH-probe EBU338 was used to set up criteria for design.
Process[edit]
- Set up criteria on Primer-3KZ program (Length=18nt, Tm=55C, GC%=40~70).
- Pick up candidate probes.
- Check the specificity (Mega-blast to nt database). Check secondary structure.
- Label with FAM on 5'-end.
- Order them on IDT at 100nmole scale with standard de-salting purification.
File:Probe design 052510.doc
Discussion[edit]
- I have concerns about the signal intensity. The unamplified DNA only, the binding of one probe won't provide enough "visialbe fluorescent emission"
- I will try to label target with combined probes.
- I also designed the Biotin-C12-probes for TSA-FISH, but they haven't been ordered yet, becaus the FAM-probe is much cheaper.