Haynes Lab:Notebook/CRISPR Editing/2016/02/23

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02/23/2016 Test qPCR primers for ChIP Dilute Gal4-EED 1 sonicated and column purified DNA from ChIP 1:100 Each reaction 1ul diluted template 1ul F primer 1ul R primer 7ul water 10ul Taq 2x MM Tm 57 Tube F primer R primer amplicon length Tm for taq 1 P313 P311 186 54 2 P309 P160 171 54 3 P309 P312 175 56 4 P308 P160 202 54 5 P308 P311 171 54 6 P314 P318 205 47 7 P316 P317 169 48 8 P315 P318 197 47 9 P316 P318 160 47 10 P316 P214 196 48 Cut MV13-KAH60 Plasmid: MV13-KAH60 6 297ng/ul, confirmed by Cass Test SgrAI 1ul SgrAI 1ul Fastdigest buffer or Cutsmart Test XbaI 1ul XbaI 1ul Cutsmart buffer or Fastdigest 2ul plasmid, 10ul total rxn Incubate at 37 for 1 hour Confirm on gel SgrAI destroyed the backbone. Need to cut with more vector, less enzyme and for a shorter time. XbaI cuts in cutsmart, SgrAI cuts in FD buffer. Western primary stain Make or thaw more blocking buffer (5% BSA, 1%PBS, 1% tween, bring to volume with DI water from tap). Get blocked nitrocellulose membranes from 4deg room. 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, 4ul each into 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. Antibodies H3-ab1791 Rb pAb from abcam New - Millipore rabbit poly 07-449 (purchased this month) Old - Millipore rabbit poly 07-449 (purchased last year)