Matt:LabNotes/2013-9-25
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Expected number of ACTB and RAB7A Rolonies
- Initial trial of secondary gene rolony generation showed low number of rolonies http://genome-tech.ucsd.edu/LabNotes/index.php/Hosuk:LabNotes/2013-9-12#Number_of_Rolonies
- I want to look at literature to see how many rolonies of ACTB and RAB7A we should expect
- http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM313643 is gene expression data for PGP1F cells using Illumina Ref-8 v3.0 beadchip (targets 18,631 unique genes)
- The table at the end of http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL6883 can be used to convert gene symbol to ILMN ID Ref
' | ID Ref | ID Ref | Expression Value | Expression Value | % of total |
ACTB | ILMN_2152131 | ILMN_2038777 | 42,723.45 | 33,579.69 | 0.32 |
RAB7A | ILMN_1716524 | N/A | 7,254.71 | N/A | 0.030 |
All Transcripts | N/A | N/A | 23,976,360 | N/A | 100 |
- According to this: http://bfg.oxfordjournals.org/content/8/6/437.full.pdf, 95% of RNA is housekeeping ncRNA. In other words ~5% of our primary rolonies is mRNA
- Therefore expect 0.016% of primary rolonies to be ACTB secondary rolonies
- Expect 0.0015% of primary rolonies to be RAB7A secondary rolonies
Dye-coupling aminoallyl-dUTP
Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-9-24
- Following Dan's Dye Coupling Protocol and Dan's Column Size Separation Protocol
- 3 5ul samples: +aadUTP, -aadUTP, and NTC (just water)
1. Warm DMSO and sodium bicarbonate buffer to RT 2. Added 3ul of of sodium bicarbonate to 5ul sample 3. Denatured samples at 95C for 5 min and then snap cooled 4. Dissolved 1 vial of Alexa 564 dye in 6ul DMSO; vortexed and spun down 5. Added 2ul of dye to each sample 6. Incubated in dark for 1 hr 7. Added 5ul 3M NaOAc and 40ul nfH2O to sample 8. Column purification
filtered + 0.5 pmol/ul; 20.3 ng/ul - 0.0 pmol/ul; 25.5 ng/ul ntc 0.0 pmol/ul; 0.5 ng/ul
non-filtered + 17.7 pmol/ul; 14.7 ng/ul - 16.3 pmol/ul; 13.9 ng/ul ntc 17.8 pmol/ul; 14.3 ng/ul (18.2; 15.2)