BAC and Fosmid DNA Isolation using 96 deep well, blocks, pipetting on the Hydra 96, and growth in the HiGro Shaker for 454 sequencing

As modified by Fares Najar in the Roe Lab at the University of Oklahoma ACGT

Updated December 15, 2009

 

1.    Pick a smear of colonies and inoculate 3 ml LB medium containing the appropriate antibiotic.

2.    Grow the culture overnight at 37oC by shaking at 250 rpm in the Floor Shaker for 8-10 hours to produce a "starter culture".  For multiple projects, this step can be accomplished using the 96-deep well block [Beckman (cat#140504)].

3.    Add 1 ml of LB medium containing the appropriate antibiotic to each well in a 96 deep well block [Beckman (cat#140504)].

4.    Inoculate 15 ul of the starter culture into each well using the Eppendorf repeater pipette or the Hydra if the starter culture is in a 96-deep well block with 8 deep wells per BAC*.

5.    Grow the BAC clone for 8-10 hours at 37oC shaking at 500 rpm in the HiGro.

6.    Centrifuge the blocks at 3000 rpm for 10 minutes. Discard the supernatant.

7.    Add 40 ul of 10mM EDTA to each well using the Hydra .

8.    Incubate on a table-top shaker for 10 minutes at speed of 7.

9.    Using the Hydra, add 80 ul of lysis buffer (1% SDS, and 0.2 N NaOH) to each well. Incubate at room temperature WITHOUT AGITATION OR SHAKING  until a clear lysate is developed.  It takes approximately 3 - 5 minutes for the clear lysate to develop.

10. Add 60 ul 3M potassium acetate (KOAc), pH 4.5 to each well using the Hydra.

11. Incubate on ice for 30 minutes WITHOUT AGITATION OR SHAKING

12. Freeze the blocks at -70oC overnight.

13. Remove the blocks from freezer and thaw. Centrifuge at 4250 rpm for 45 mins in the Jouan KR422.

14. Transfer 130 ul of the supernatant from each well to a new block using the Hydra

15. Add 130 ul of isopropanol to each well using the manual Eppendorf repeater pipette.

16. Centrifuge at 4250 rpm for 45 mins in the Jouan KR422 to collect DNA. Decant the supernatant. Invert on a paper towel.

17. Wash with 500 ul 70% ethanol and centrifuge at 3000 rpm for 10 mins in the Jouan KR422.  Air-dry for 10 minutes.

18. Dissolve the pellet in 100 ul of TE-RNaseA/T1 using the Hydra followed by shaking on a table top microtiter shaker at speed of 5 for 5 minutes to dissolve the DNA.

19. Add 50 ul of 7.5 M of KOAc*** using Eppendorf repeater pipette and freeze at -80oC overnight.

20. Remove the blocks from the freezer and thaw.  Centrifuge at 4250 rpm for 45 mins in the Jouan KR422.

21. Transfer 100 ul of the supernatant using the Hydra.

22. Add 100 ul of isopropanol and centrifuge at 4250 rpm for 45 mins in the Jouan KR422..

23. Wash with 500 ul 70% ethanol twice and dissolve the DNA in 20 ul sdd-water.

24.  Typically the yield is about ~5 ng/ul final 20 ul solution or 100 ng / well (800 ng/8 wells).

 

Notes:

* We typically inoculate all 8 wells in a column with a single BAC and 12 BACs per 96 well plate.

** TE-RNaseA/T1 is 40 ug/ml RNase A and 40 U/ml RNase dissolved in 50:10 TE buffer where DNase-free RNase A was diluted from a 20 mg/ml RNase A stock solution in 1 mM NaOAc, pH 4.5; Sigma R-5500 and 40 U/ml RNase T1 was diluted from a 100 U/ul iRNase T1 stock solution in 50 mM Tris-HCl, pH 7.6; Sigma R-8251**

***7.5 M KOAc Stock Solution: 736.13 gm/liter final volume - Starting with a small volume of dd-water (approximately150ml) slowly add the dry potassium acetate and mix as it is added. Then add additional water and the remainder of the acetate until all is dissolved and the final volume is 1 liter. DO NOT ADJUST THE pH as the acetate will come out of solution.