Haynes Lab:Notebook/CRISPR Editing/2015/03/06

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03/06/2015 miniprep of pMax GFP 6.2 from Lonza and KAH60 plasmid colony 260 280 260/280 ng/µL ul for 05.ng pMax GFP 6.2 1 0.552 0.299 1.848 551.779 0.906159894 pMax GFP 6.2 2 0.348 0.187 1.861 347.553 1.438629504 KAH60 1 0.102 0.056 1.817 101.806 4.911301888 KAH60 2 0.278 0.148 1.878 278.411 1.795906053 plasmid ng/µL ul for 05.ng g044 403.212 1.240042459 g031 288.187 1.734984576 plated cells look great, all wells are about 90% confluent. Will proceed with all 48 transfections KAH60 transfections Step 1 ' ' ' ' Making 1ug/10ul plasmid stocks ng/ul ul plasmid ul H2O KAH60 278.411 51.00 91.00 pMax GFP 6.2 551.779 14.86102226 75.13897774 g032 452.739 13.47 48.53 g031 288.187 21.17 40.83 g025 235.95 25.85 36.15 g044 403.212 15.13 46.87 Step 2 make DNA + optimem mixes KAH60 add 554 ul optimem pMax GFP 6.2 add 320 ul optimem g032 add 242 ul optimem g031 add 242 ul optimem g025 add 242 ul optimem g044 add 242 ul optimem Step 3 add plus reagent to DNA+ opti KAH60 add 14.2ul plus pMax GFP 6.2 add 8.2ul plus g032 add 6.2ul plus g031 add 6.2ul plus g025 add 6.2ul plus g044 add 6.2ul plus step 4 Make optimem + lipo mastermix Optimem 2265.4 lipo 144.6 Step 5 mix dna mix and lipo mix KAH60 add 710 lipo mix pMax GFP 6.2 add 410 lipo mix g032 add 310 lipo mix g031 add 310 lipo mix g025 add 310 lipo mix g044 add 310 lipo mix Step 6 add 100ul dropwise to each well