(No Exonuclease treatment)
Uses 200 ml LB culture

1. Streak out BACs from glycerol stock and grow overnight at 37°C on LB with antibiotic of choice (Qiagen recommends LB of 10g Tryptone, 5g Yeast Extract, 10g NaCl).

2. Using a single colony the next day, inoculate 2 ml LB/anitbiotic (use sterile pipette tip, and just drop it into the starter culture). Grow starter culture approximately 8 hours.

3. After 8 hours, use approximately 200µL of starter, to inoculate a 200ml LB/antibiotic culture (in 1L flask). Grow at 37°C for 12-16 hours.

4. Harvest cells in 240 ml bottles. Pellet cells by spinning tubes in large GSA rotor (holds 6 tubes) at 6000xg for 15 minutes at 4°C. Pour off supernatant.

For the next steps, gentleness is key, so that isolation of bacterial genomic DNA is limited!

5. Add 10ml chilled buffer P1 to pellet and gently resuspend pellet using electronic pipette

(for next step I usually process 2-3 bottles at a time and keep the rest on ice. The lysis should not proceed any longer than 5 minutes--this is key).

6. Set a timer for 5 minutes before adding 10 ml buffer P2 down the sides of the bottles, as not to agitate the cells. Mix very gently by rotating bottles once 360° and laying the bottles on their sides for 2 minutes. After two minutes, rotate bottles again 360° and set bottles up on end for two more minutes.

7. At the end of two minutes, add 10ml ice-cold buffer P3 by gently pouring down the sides of the bottle, and gently rotate the bottles 360° twice. Set on ice for 15 minutes.

8. Carefully transfer mixture to smaller, 35 ml Sorvall tubes (keep on ice). Change to smaller rotor, and spin down cell debris at >20,000g for 15 min. at 4°C. Pour supernatant immediately into a new Sorvall tube and spin again for 15 minutes to get rid of remaining debris.

9. After the 2^nd spin, pour supernatant into a third Sorvall tube, by filtering the supernatant through a coffee filter lined funnel. Squeeze the filter to get as much supernatant out as possible.

10. Measure volume of supernatant with a 25ml glass pipette. Add 0.7 of this volume of ispropanol to each tube, mixing gently. Centrifuge at >20,000g, 4°C for 30 minutes.

11. Pour of supernatant. Gently add 5 mls of 80% ETOH to each tube, and carefully rotate tube so that the ethanol bathes the whole tube. Spin tube for 15 more minutes.

12. After spin. gently pour off ethanol, and sit tubes upside down on a paper towel for a minute or two, to get rid of remaining ethanol. Then lay tubes on side for several minutes, so that remaining ethanol evaporates. NOTE: do not overdry pellets here, or the BAC DNA will not go back into solution! It's better to leave the pellet a little "wet" than to over dry.

13. Add 400µL T10E1 buffer to each pellet. Gently shake the liquid over the pellet to get the DNA in solution--do not pipette up and down, or you will shear the BAC DNA. Sit the tubes on their side for 15 minutes, to ensure all the DNA is in solution.

14. Add 2 mls buffer QBT to each sample. There will likely still be some white "junk" that does not go into solution at this step. To get rid of it, spin tubes for 10 minutes at >20,000g (4°C).

During this spin, pre-equilibrate each Qiagen-20 column with 1 ml buffer QBT.)

15. After the spin, remove supernatant from each tube and load it on the column, taking care to not load any white junk on the column. Allow the supernatant to enter the column by gravity flow.

(While the sample is entering the column, aliquot some elution buffer (buffer QF) to a tube and heat at 65°C.)

16. Wash the samples twice with 2ml buffer QC.

17. After was buffer has gone through the columns, elute the DNA into labeled eppendorf tubes two times with 400µL (total 800µL) heated elution buffer QF).

18. Precipitate DNA with 0.7 volumes room temp isopropanol (0.56ml). Gently mix tubes and spin down DNA for 30 minutes at 14,000g.

19. Remove supernatant and wash with 1 ml 70% ethanol. Spin down 5 minutes at 14,000g. Remove all ethanol and dry pellets briefly. NOTE: Do not over dry the pellets, or the DNA will not go into solution!

20. Very gently resuspend pellet in 100µL T10E1.

21. Incubate samples at 65°C for 30 minutes to ensure all DNA is in solution. After incubation, gently pipette DNA one or two more times. 22. Store at 4°C.