Matt:LabNotes/2013-9-25

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Expected number of ACTB and RAB7A Rolonies[edit]

' 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[edit]

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 15ul H2O to dye-coupled samples to make 25ul total
 8. Took 0.5ul of dye-coupled sample and diluted to 10ul for non-filtered measurements
 9. Added 24.5ul of sample to prepped Centri-Sep column and spun for 2 min at 750rcf
 10. Measured dye conc and DNA conc using Nanodrop Microarray setting (dsDNA)
' ' Alexa 564 (pmol/ul) DNA conc (ng/ul) DNA (pmol/ul) Bases (pmole/ul) Base:Dye Ratio
Filtered +aadUTP 0.5 20.3 0.346074442 62.29339953 124.5867991
Filtered -aadUTP 0 25.5 0.434724053 78.25032945
Filtered NTC 0 0.5 0.008524001 1.534320185
Nonfiltered +aadUTP 17.7 14.7 0.25060563 45.10901345 2.548531833
Nonfiltered -aadUTP 16.3 13.9 0.236967229 42.65410115 2.616816022
Nonfiltered NTC 17.8 14.3 0.243786429 43.8815573 2.465256028
  • 90bp DNA mass: 58,658 g/mol
  • The Nonfiltered NTC is messed up, possibly could've taken the 0.5 ul from wrong tube in step 8
  • Among the filtered samples, good evidence that the aadUTP is still good
    • Base:Dye ratio isn't the best but definitely falls within the range that Dan gets Daniel:ProbeList
    • He also uses 3x more dye for a reaction which could explain why I got less dye/base