Haynes Lab:Notebook/Engineering PC-TFs/2014/03/22

Main project page Previous entry      Next entry

Summary Miniprep on ligated plasmid CMV MV9 liquid culture. Gel purification from prior CMV miniprep. Performed mini-prep on each of the four picked colonies. Ran concentration check on each: Plasmid OD260 OD260/280 ng/μL 1. Colony 1 0.005 1.211 4.895 2. Colony 2 0.003 1.273 2.963 3. Colony 3 0.034 1.888 33.572 4. Colony 4 0.007 -5.308 7.215 Digest CMV Restriction Digest Table Checked plasmid minipreps with EcoRI/PstI digests Reagent Volume Expected: 1. CMV = 588bp DNA(CMV) 25.0 μL 10X buffer 3.0 μL EcoRI 1.0 μL PstI 1.0 μL dH2O 0 μL   15 μL --> 37°C/ 15 min. Gel Purification: CMV/V0120 Using Prior Miniprep Digests CMV culture 1 - check with E/P Used previously digested CMV. Placed 15μL 1kB Ladder in well one and 30μL of CMV digest in well two. Ran gel at 110V for 30 minutes. Placed gel on UV transilluminator and cut out desired bands. 15 μL/lane; 1% agarose; cut the brightest band furthest down at ~ 588bp; Ladder Used Zymo gel purification kit. Assumed 200mg gel, added 600μL ADB to each 1.5mL tube. Put on heat block at 55°C for 5 minutes, vortexed and then placed back on the heat block for an additional 5 minutes, vortexed again. Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds. Pipetted 200μL wash buffer to each tube, centrifuged at max rpm for 30 seconds. Repeated once. Transferred columns to new labeled 1.5mL tube. Pipetted 15μL elution buffer to each column. Spun at max rpm for 30 seconds. Put marked (CMV gel pur2 3/22) CMV in -20°C fridge in box labeled Cameron.