User:Karmella Haynes/Notebook/PcTF Genomics/2015/08/18: Difference between revisions

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Run gel: 120 V, 60 min.
'''TRANSFER'''
Electroblot
* Load both at same time, "feet" facing inward
* Transblot Turbo transfer pack, mini format, 0.2 um PVDF
* Bio-Rad Transblot Turbo: Bio-Rad Protocol, Mixed MW, 2.5A, 25V, 7 min (slot A, upper)
Poncaeu stain
* Rinsed blot in di-water and wahed in 1x Poncaeu for ~15 min.
* Rinsed off excess with di-water
RESULTS




Revision as of 11:43, 18 August 2015

Pc-TF Genomics <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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08/18/15

  • Western - Castone PAGE & blot


Western - Castone PAGE & blot

  • Troubleshooting: saw some indication of signal last time; BSA is recommended for modified proteins
    • Re-run the Gal4-EED sample set
    • Use BSA-based blocking buffer for downstream steps


SDS-PAGE

General:

Protein sample prep:

  • Final volume = 20 μL
  • Sample buffer: 4x Nupage LDS sample buffer
  • 4x sample master mix (x13)
    • 13x 5.0 μL 4x LDS = 125
    • 13x 1.0 μL 1M DTT = 25
  • Protein samples (6): 14 μL protein + 6 μL master mix
  • Dummy samples (5): 14 μL water + 6 μL master mix
  • Heat @ 100°C/ 5 min.Cool at room temp.

Loading:

  1. PageRuler Plus
  2. Luc14: H340_MV11 + g034_pSPgRNA
  3. Luc14: H340_MV11 (no gRNA)
  4. Luc14: H360_MV11 + g034_pSPgRNA
  5. Luc14: H360_MV11 (no gRNA)
  6. Luc14: H380_MV11 + g034_pSPgRNA
  7. Luc14: H380_MV11 (no gRNA)
  8. dummy
  9. dummy
  10. dummy
  11. dummy
  12. dummy

Run gel: 120 V, 60 min.


TRANSFER

Electroblot

  • Load both at same time, "feet" facing inward
  • Transblot Turbo transfer pack, mini format, 0.2 um PVDF
  • Bio-Rad Transblot Turbo: Bio-Rad Protocol, Mixed MW, 2.5A, 25V, 7 min (slot A, upper)

Poncaeu stain

  • Rinsed blot in di-water and wahed in 1x Poncaeu for ~15 min.
  • Rinsed off excess with di-water


RESULTS




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