Haynes Lab:Notebook/CRISPR Editing/2016/02/29: Difference between revisions
Rene M Davis (talk | contribs) |
Rene M Davis (talk | contribs) |
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Make or thaw more blocking buffer (5% BSA, 1%PBS, 1% tween, bring to volume with DI water from tap). | Make or thaw more blocking buffer (5% BSA, 1%PBS, 1% tween, bring to volume with DI water from tap). | ||
<br> | <br> | ||
Make parafilm pouches! Cut a strip of parafilm that can be folded over to make a pouch just a few millimeters wider than each membrane. Lay the membrane in the unmade pouch and make the pouch around the membrane by smooshing double-folded parafilm edges with the spatula tool. leave the top open, pipette in 1mL blocking buffer into the pouch, set aside and watch for leaks. Make the other pouches the same way. Dilute the antibodies, | Make parafilm pouches! Cut a strip of parafilm that can be folded over to make a pouch just a few millimeters wider than each membrane. Lay the membrane in the unmade pouch and make the pouch around the membrane by smooshing double-folded parafilm edges with the spatula tool. leave the top open, pipette in 1mL blocking buffer into the pouch, set aside and watch for leaks. Make the other pouches the same way. Dilute the antibodies '''need to look up how much i added, v imp!''' 1mL blocking buffer. Confirm that none of the pouches have leaked. Add the antibodies to the appropriate pouches, LABEL each pouch with the antibody. Seal the tops, rotate at 4degC overnight. <br> | ||
Antibodies | Antibodies | ||
:H3-ab1791 Rb pAb from abcam | :H3-ab1791 Rb pAb from abcam |
Revision as of 08:11, 13 March 2016
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02/29/2015Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow
insert calculations for 1:2 ratio
Mixed gently, inc at RT for 30 mins
Ligation for MV14
Insert
Mixed gently, inc at RT for 30 mins 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)
Cut the nitrocellulose to separate the pieces that will be bound with different antibodies. add notches to one corner so you remember the orientation
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