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AGI > Research > Protocols > BAC DNA Prep for Shotgun Library Construction

BAC DNA Prep for Shotgun Library Construction

1. From a plate or stab tube, make a 3ml culture with 2XYT + CM (12.5ug/ml) and incubate in 37 C, shaking at 250rpm for 4 hours.

2. Transfer 50ul of culture to a 50ml flask with 50ml 2XYT + CM (12.5ug/ml), and incubate/shake for 14-16 hours at 250rpm. (14 hour incubation best)

3. Prepare all solutions and reagents needed for DNA prep.

4. Transfer culture in each flask to two 50ml centrifuge tubes, dividing the sample evenly between the tubes.

5. Centrifuge tubes at 9,000 rpm for 10 minutes at 4 C (green line should be set to ~13 C).

6. Allow tubes to drain upside down on paper towels.

7. Place 2ml of Solution 1 (10mM EDTA ) into each tube.

To make: 1ml of 0.5M EDTA
49ml ddH2O ~10 samples

8. Place cap back on and vortex until no pellet left.

8. Add 4ml Solution 2. To make: First add about 30ml ddH2O
Then 2.5ml of 4N NaOH ~5 samples
Then 5ml of 10% SDS
Bring remaining volume up to 50ml with ddH2O

9. IMMEDIATELY add 3ml of Solution 3. KEEP SOLUTION 3 COLD. Keep tubes on ice as you go along.
To make Sol. 3: 12.5ml 7.5M KOAc
5.75ml of Acetic Acid ~8 samples
31.75ml of ddH2O

10. Centrifuge for 15 min. at 12000 rpm.

11. Remove tubes CAREFULLY and put on gloves.

12. Filter using the larger miracloths, handling with forceps.

13. Repeat step 10.

14. Filter again using the smaller miracloths.

15. Add 9ml isopropanol to each tube and invert well.

16. Centrifuge 15 min. at 12000 rpm.

17. Pour out supernatant and keep the remaining pellet.

18. Drain tubes upside down on paper towels.

19. Dissolve pellet with 2.25ml T10E50 (10mM Tris and 50mM EDTA).
To make 10ml: 100ul 1M Tris
1ml 0.5M EDTA
8.9ml ddH2O ~4 samples

20. Tap tubes with finger to mix.

21. Add 1.15ml of 7.5M KOAc, taping tubes to mix.

22. Lay tubes on their side on a paper towel in -80 C for at least 30 minutes.

23. Allow tubes to thaw 10-15 minutes.

24. Immediately centrifuge at 6500 rpm for 10 minutes.

25. Filter with the smaller miracloths as before.

26. Add 8.5 ml of 95% EtOH and mix well.

27. Centrifuge at 12,000 rpm for 15 minutes.

28. Pour off supernatant and place tubes upside down on paper towels.

29. Dry pellet in vent room for 5 minutes.

30. Dissolve pellet in 700ul T50E50, taping tube with finger.
To make: 500ul 1M Tris
1ml 0.5M EDTA
8.5ml ddH2O

31. Quickspin to 5000 rpm.

32. Add 5ul RNase (Roche Brand!)

33. Incubate tubes for an hour, shaking at 50 rpm in 37 C.

34. Transfer samples to labeled eppendorf tubes.

35. Put on gloves.

36. Add 600ul of phenol, avoiding the top layer.

37. Vortex tubes until there is only one mixture.

38. Centrifuge in 4 C refrigerator at 13000 rpm for 5 minutes.

39. Transfer the top phase of the sample to a new eppendorf tube.

40. Add 500ul of phenol.

41. Repeat steps 37 through 39, being EXTREMELY careful to only pipett the top layer.

42. Add 700ul of isopropanol.

43. Vortex and centrifuge at 13000 rpm for 30 minutes.

44. Pour off supernatant.

45. Add 500ul of 70% EtOH.

46. Centrifuge at 13000 rpm for 15 minutes.

47. Pour off supernatant and dry tubes in the vent room for 10-15 minutes.

48. Re-suspend pellet in 40ul T10E50.

49. If not using sample that day, store in -20 C.
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A portion of AGI's material is based upon work supported by the National Science Foundation under Grant Number 102620.