Matt:LabNotes/2013-9-25: Difference between revisions

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(Created page with "===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.p...")
 
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*Therefore expect 0.016% of primary rolonies to be ACTB secondary rolonies
*Therefore expect 0.016% of primary rolonies to be ACTB secondary rolonies
*Expect 0.0015% of primary rolonies to be RAB7A 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 [[Daniel:Protocols/AlexaDyeCouple | Dan's Dye Coupling Protocol]] and [[Daniel:Protocols/CentriSep | 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

Revision as of 22:21, 25 September 2013

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