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Miniprep Protocol 9/23/13 TO
Total Time: 1 day to grow up the plasmid and about 1 hour to miniprep. Purpose: The purpose of the miniprep protocol is to isolate plasmids from cells. We can then use these plasmids to perform any number of operations from PCR, to transformations. We keep miniprepped plasmids in the -‐20 freezer in the “Lab Plasmid Stock” box. Check this box to see if your plasmid is already miniprepped. 1.) Grow up a patch of the plasmid you want to isolate from either the -‐80 stocks or a transformation you have completed yourself. 2.) From the patch (or colony) take a swipe of cells with a pipette tip. Colony will give you about 115 ng/μl while patch will give you about 300 ng/μl.
3.) Eject this tip directly into a 5ml tube that has 3mls of LB + Antibiotic. (We add 1μl of a 100 ng/μl of Amp per 1ml of LB.) 4.) Grow this up overnight at 37° shaking at about 200-‐230 rpm. 5.) After growth phase, remove 1.5 mls into the provided centrifuge tube. Centrifuge for 1 min at 13,000 rpm. 6.) Descant the fluid and tap on a clean paper towel. 7.) Add the remaining 1.5 mls into that same centrifuge tube. Spin for another min at 13,000 rpm. Descant the fluid and tap on clean paper towl. 8.) Resuspend cells in 400μls of COLD LYSIS BUFFER. Vortex at least 30 seconds until all cells are in solution. Make sure cells are not in pellet at the bottom. This step is critical to get good yield. 9.) Incubate at Room Temp for 5 min.
Colony Patch
10.) Centrifuge 1 min. at 13,000 rpm 11.) Add 400μl of Wash Buffer 12.) Centrifuge 1 min. at 13,000 rpm 13.) Dump flow through and tap on clean paper towel. 14.) Centrifuge for an additional 1.5 min at 13,000 rpm to dry the filter. 15.) Transfer filter to a new GeneMate 1.5μl microcentrifuge tube. Add 50μl of Elution Buffer to each sample. 16.) Let incubate at room temperature for 5 min. 17.) Centrifuge for 1 min. at 13,000 rpm. You MUST bend back the cap on the microcentrifuge tube so that it is flat, or else the cap will break off. The GeneMate brand tend to break less than the FastPlasmid brand. 18.) Nanodrop your samples. A miniprep done from a colony will yield about 113 ng/μl whereas a minipred done from a patch will yield about 300 ng/μl.