Mini-prep
Notes before starting
- Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000
- Add the provided RNase A solution to Buffer P1, mix, store bottle at 2-8C
- Add ethanol (96-100%) to Buffer PE before use
- All centrifugation steps are carried out at 13,000 rpm (~17,900xg) in a conventional table-top microcentrifuge
Mini-prep
- Centrifuge 1-6mL bacterial overnight culture at >8000 rpm (6800xg) for 3 minutes at room temperature (15-25C)
- Resuspend pellet in 250ul Buffer P1 and transfer to microcentrifuge tube
- Add 250ul Buffer P2 and mix thoroughly by inverting the tube 4-6 tiems until the solution becomes clear.
- DO NOT allow lysis reaction to proceed for more than 5 minutes
- If using LyseBlue reagent, the solution will turn blue
- Add 350ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
- If using LyseBlue reagent, the solution will turn colorless
- Centrifuge for 10 minutes at 13,000 rpm (~17,900xg) in a table-top microcentrifuge
- Apply supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through
- Recommended: Wash the QIAprep spin column by adding 500 ul Buffer PB. Centrifuge for 30-60 s and discard the flow-through
- Only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content
- Wash the QIAprep spin column by adding 750 ul of Buffer PE. Centrifuge for 30-60 s and discard the flow-through
- Centrifuge for 1 minutes to remove residual wash buffer
- Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 30ul Buffer EB. Let stand for 1 min, and centrifuge for 1 minute
- Could elute DNA in 50ul
- To increase yield, let sit for up to 4 minutes
Protocol was modified from the Qiagen Mini-prep kit instructions
