Matt:LabNotes/2013-9-25

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Revision as of 23:30, 25 September 2013 by >Mzcai (→‎Dye-coupling aminoallyl-dUTP)
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Expected number of ACTB and RAB7A Rolonies

' 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

 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)