Qiagen kit miniprep

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
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
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