Qiagen kit miniprep From Qiagen manual 1. Streak E. coli containing your plasmid(s) on selective media. Grow o/n at 37°. 2. Pick ONE colony and inoculate a 2-5ml culture(s) of selective media. Grow o/n at 37°. 3. Decant culture (s) into an Eppendorf tube and microfuge 30 sec. to pellet your cells. Discard supernatant. 4. Resuspend pellet in 250µl Buffer P1 (in the 4°C fridge) by scraping 5-10 times along an Eppendorf rack. No cell clumps should be visible at this point. 5. Add 250µl Buffer P2 (lysis buffer) and immediately but gently invert 4-6 times to mix. Do not vortex! Solution will become clearer. Do not allow the lysis reaction to proceed longer than 5 min. 6. Add 350µl Buffer N3 and invert the tube and immediately but gently invert 4-6 times to mix. You should get clumps of white gunk at this point – if the solution does not become clumpy you are probably shaking the tube too hard. 7. Microfuge 10 min. (If you are going to run the miniprepped plasmid on a gel, now is a good time to set up the gel). 8. Decant the supernatant into a QIAprep spin column, ensuring that no white gunk has gone with it. Discard the tube. 9. Centrifuge 30-60 sec. and discard flow-through. 10. Wash the column with 750µl Buffer PE. Centrifuge 30-60 sec. and discard flowthrough. 11. Centrifuge for an additional 1 min. to remove residual Buffer PE. IF YOU DO NOT FOLLOW THIS STEP YOUR DNA WILL NOT WORK AS WELL. 12. Move the QIAprep column to a clean, labeled Eppendorf tube. Apply 50µl of Buffer EB (elution buffer) to the column – it is important that you get the EB in the center of the column. Allow to sit 1 min., then centrifuge 1 min. 13. Use 1-3µl miniprep for diagnostic gel and freeze remainder of plasmid DNA. To make a DNA gel: Mix 0.5g agarose with 60ml TAE. Microwave (checking every 30 sec. or so) until all agarose is dissolved. Allow to cool in 55°C waterbath while you prepare the gel mold. Add 5-6µl EtBr immediately before pouring. Allow gel to solidify. Mix plasmid with 6X DNA sample buffer and load into gel wells. Run at 60-70mA and observe bands under UV.