User:Karmella Haynes/Notebook/PcTF Genomics/2015/08/12

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08/12/15

  • Castone Western - gel and transfers

Castone Western - gel and transfers

  1. Luc14: H340_MV11 + g034_pSPgRNA
  2. Luc14: H340_MV11 (no gRNA)
  3. Luc14: H360_MV11 + g034_pSPgRNA
  4. Luc14: H360_MV11 (no gRNA)
  5. Luc14: H380_MV11 + g034_pSPgRNA
  6. Luc14: H380_MV11 (no gRNA)
  7. Gal4EED-luc: H340_MV11 + g034_pSPgRNA
  8. Gal4EED-luc: H340_MV11 (no gRNA)
  9. Gal4EED-luc: H360_MV11 + g034_pSPgRNA
  10. Gal4EED-luc: H360_MV11 (no gRNA)
  11. Gal4EED-luc: H380_MV11 + g034_pSPgRNA
  12. Gal4EED-luc: H380_MV11 (no gRNA)


SDS-PAGE

General:

Protein sample prep:

  • Final volume = 20 μL
  • Sample buffer: 4x Nupage LDS sample buffer
  • 4x sample master mix (x25)
    • 25x 5.0 μL 4x LDS = 125
    • 25x 1.0 μL 1M DTT = 25
  • Protein samples (12): 14 μL protein + 6 μL master mix
  • Dummy samples (10): 14 μL water + 6 μL master mix
  • Heat @ 100°C/ 5 min.Cool at room temp.

Loading - Gel 1:

  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

Loading - Gel 2:

  1. PageRuler Plus
  2. Gal4EED-luc: H340_MV11 + g034_pSPgRNA
  3. Gal4EED-luc: H340_MV11 (no gRNA)
  4. Gal4EED-luc: H360_MV11 + g034_pSPgRNA
  5. Gal4EED-luc: H360_MV11 (no gRNA)
  6. Gal4EED-luc: H380_MV11 + g034_pSPgRNA
  7. Gal4EED-luc: H380_MV11 (no gRNA)
  8. dummy
  9. dummy
  10. dummy
  11. dummy
  12. dummy

Run gels: 120 V, 50 min.

  • Note: next time run 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)
  • Note: Wow! worked super well and only took 7 min! However, did not see 10 kD band on the membrane, but 15 kD is there (histones)

Poncaeu stain

  • Rinsed blot in di-water and wahed in 1x Poncaeu for ~15 min.
  • Rinsed off excess with di-water
  • Note: Pxi4 imager not set up correctly, troubleshoot tomorrow with Solo Pyon


08/13/15 - RESULTS

Blot 1 - Luc samples 1-6
Blot 2 - Gal4EED-luc samples 7-12