Haynes Lab:Notebook/CRISPR Editing/2016/02/29: Difference between revisions
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==02/29/2015== | ==02/29/2015== | ||
Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow<br><br> | |||
'''Running gel for western'''<br> | '''Running gel for western'''<br> | ||
Got NuPAGE LDS Sample buffer 4x out of 4deg fridge<br> | Got NuPAGE LDS Sample buffer 4x out of 4deg fridge<br> | ||
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run gel for 1 hour at 120V (until lowest dye band ran just past the third ridge in gel box) | 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 [https://benchling.com/hayneslab/f/8iVwOlNf-protein-and-enzyme-assays/prt-fiQyr0E7-western-blot/edit protocol].<br> | |||
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. <br> | |||
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. | |||
<br> | |||
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.<br> | |||
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.<br> | |||
Cut the nitrocellulose to separate the pieces that will be bound with different antibodies. add notches to one corner so you remember the orientation<br> | |||
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) | |||
Revision as of 09:30, 29 February 2016
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02/29/2015Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow Running gel for western Gel lanes
Mastermix for sample buffer
heat at 100degC for 5 mins, cool in rack at RT
remove the strip from the gel, put premade gel into box with the open part facing the outer chamber, fill inner chamber to cover the comb, outer chamber to cover the bottom of the gel.
run gel for 1 hour at 120V (until lowest dye band ran just past the third ridge in gel box)
we had trouble getting it to start, had to move to B.
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