Haynes Lab:Notebook/CRISPR Editing/2016/02/22

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02/22/2016

Ran an SDS protein gel with KAH following this protocol

Gel lanes

  1. stock: ladder
  2. sample prep: Luc14 1 (14 uL protein)
  3. sample prep: Luc14 2 (14 uL protein)
  4. sample prep: dummy (14 uL water)
  5. " ladder
  6. " Luc14 1
  7. " Luc14 2
  8. " dummy
  9. " ladder
  10. " Luc14 1
  11. " Luc14 2
  12. " dummy


Samples were prepared Friday
Ladder found in -20 protein reagents box

Gel/ Buffer

  • NuPAGE 4-12% BisTris
  • NuPAGEĀ® MOPS SDS Running Buffer


Sample prep

  • 5 ul 4x Loading Buffer, 1 uL 10M DTT, 14 uL protein or water
  • heat at 100degC for 5 mins, cool in rack at RT


prep gel

  • put premade gel into box, fill inner chamber, outer chamber.
  • load samples and ladder
  • run gel for 1 hour at 120V (until lowest dye band ran just past the third ridge in gel box)


Transfer and blocking following this protocol.

Remove the gel from the box, rinse with DI water, scrape the exposed gel slit on the bottom to get all the gel from that spot. Rinse the gel gunk away. Crack the plastic apart but don't pull it apart until the plastic is unstuck. With the gel submerged in a tupperware filled with DI water, separate the plastic pieces. Pay attention to which plastic part the gel is sticking to. Pay attention to the direction of your gel. Get the gel floating in the DI water.
Transfer the gel to nitrocellulose paper using the transblot machine. Make sure to keep the orientation.

list
biorad
1 minigel
mixed mw
run
A-run

we had trouble getting it to start, had to move to B.
Should have no bands in the gel and bands on the nicrocellulose. Rinse the nitrocellulose in DI water. Move the nitrocellulose to a smaller dish. Pour Ponceau stain over to just cover it. Orbital shake gently for 5-10 minutes. Rinse the Ponceau in the sink in DI water until the background is white.
Image the nitrocellulose on the syngene PXi machine. Remove the UV box and replace with the black background piece. Blots>visible blot>Ponceau red> -> or used saved protocols "ponceau stain". Keep nitrocellulose in water until time to image. Keep water nearby so you can quickly transfer. Need to make sure it doesn't dry out. Take an image.
Cut the nitrocellulose to separate the pieces that will be bound with different antibodies. add notches to one corner so you remember the orientation
To block the nitrocellulose, move to a new dish and pour 5% BSA in 1% PBS over. Ok to stack the nitrocellulose. Keep still at RT for one hour or at 4degC overnight. (we did overnight)



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