QIAprep® Spin Miniprep Kit

Notes:

• Grow clones STBL2  @  30˚C and STBL3 @ 37˚C

Materials and Reagents

• The QIAprep Spin Miniprep Kit (cat. nos. 27104 and 27106) can be stored at room temperature (15–25˚C) for up to 12 months.

• 12 ml Falcon tubes

Equipment

• Centrifuge

  
  

Procedure

1. Grow bacteria clone (~2.5 ml) overnight in LB with appropriate antibiotic

2. 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.

3. Add ethanol (96–100%) to Buffer PE before use (see bottle label for

4. volume).

5. Transfer 2 ml of culture into a 2ml eppendorf tube

6. Pellet the bacterial overnight culture by centrifugation at >5,000 rpm for 10 min at room temperature (15–25˚C).

7. Pour the supernatant out and place Eppendorf tube in a rack

8. Remove the left over LB media that drip down onto the pellet

9. Resuspend pelleted bacterial cells in 250 μl Buffer P1

10. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution

11. becomes blue. Do not allow the lysis reaction to proceed for more than 5 min.

12. 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.

13. Centrifuge for 10 min at 13,000 rpm (~17,900 xg) in a table-topmicrocentrifuge.

14. Label the QIAprep spin column

15. Apply the supernatant from step 5

16. Centrifuge for 1 min at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge. Empty the bottom tube

17. Wash the QIAprep spin column by adding 0.5 ml Buffer PB.

18. Centrifuge for 1 min at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge. Empty the bottom tube

19. Wash the QIAprep spin column by adding 0.75 ml Buffer PE.

20. Centrifuge for 1 min at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge. Empty the bottom tube

21. Spin again for 1min

22. Dry the column by waiting 5 min

23. Transfer column to labelled Eppendorf tube

24. Add 50 ul of water

25. 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