QIAprep® Spin Miniprep Kit
Notes:
Materials and Reagents
Equipment
Procedure
Grow bacteria clone (~2.5 ml) overnight in LB with appropriate antibiotic
Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000. Add the provided RNase A solution to Buffer P1, mix, and store at 2–8˚C.
Add ethanol (96–100%) to Buffer PE before use (see bottle label for
volume).
Transfer 2 ml of culture into a 2ml eppendorf tube
Pellet the bacterial overnight culture by centrifugation at >5,000 rpm for 10 min at room temperature (15–25˚C).
Pour the supernatant out and place Eppendorf tube in a rack
Remove the left over LB media that drip down onto the pellet
Resuspend pelleted bacterial cells in 250 μl Buffer P1
Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution
becomes blue. Do not allow the lysis reaction to proceed for more than 5 min.
Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube4–6 times. If using LyseBlue reagent, the solution will turn colorless.
Centrifuge for 10 min at 13,000 rpm (~17,900 xg) in a table-topmicrocentrifuge.
Label the QIAprep spin column
Apply the supernatant from step 5
Centrifuge for 1 min at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge. Empty the bottom tube
Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
Centrifuge for 1 min at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge. Empty the bottom tube
Wash the QIAprep spin column by adding 0.75 ml Buffer PE.
Centrifuge for 1 min at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge. Empty the bottom tube
Spin again for 1min
Dry the column by waiting 5 min
Transfer column to labelled Eppendorf tube
Add 50 ul of water
Centrifuge for 1 min at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge. Empty the bottom tube
References
http://www.qiagen.com/knowledge-and-support/resource-center/resourcedownload.aspx?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en