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A. Hall, revised 2/4/2003 *This protocol is just a slight modification from the Qiagen large-construct prep This prep is useful for generating BAC DNA that is good for shotgun libraries, as it starts with 500ml culture and has an exonuclease step to get rid of E. Coli genomic DNA. Cell Growth 1. Streak out fresh colony of bacteria on selective media (starting from a very fresh plate is essential!). 2. Grow a 2ml LB (not TB) starter culture 8 hrs, 37°C at 300rpm. I follow the Qiagen LB recipe, with 10g Tryptone, 5g Yeast Extract, and 10g NaCl. 3. Dilute 500µL of the starter into 500ml LB/antibiotic. 4. Grow 16 hrs. at 37°C. 5. Pellet cells in large bottles at 6000G, 15 min. at 4°C. Note: You don't want to overfill the bottles, so it will take two spins. Cell Lysis For the next steps, gentleness is key, so that isolation of bacterial genomic DNA is limited! 6. Add 50 mls chilled buffer P1 to pellet and gently resuspend pellet using electronic pipette. (for the 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) 7. Set a timer for 5 minutes before adding the 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. 8. At the end of two minutes, add 50 mls 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. Before putting in centrifuge, gently mix bottle end over end 2X. 9. Spin down cell debris at 9,000xg for 15 minutes at 4°C (largest GS-3 rotor). Immediately transfer supernatant to smaller bottles, and do a high speed spin in the GSA rotor at >20,000xg for 15 minutes. 10. Filter lysate through a folded filter prewet with distilled water. 11. Measure lysate in a graduated cylinder and then precipitate DNA by adding exactly 0.6 volumes isopropanol. 12. Mix gently several times and centrifuge immediately at >15,000g for 30 minutes at 4°C. 13. Carefully decant supernatant. 14. Wash pellet with 5 mls 70% ethanol. Gently rotate the tube to make sure the ethanol soaks all parts of the pellet and tube. Spin >15,000g for 15 minutes at $°C. *At this step, prepare the reagents for the exonuclease digestion. Dissolve the exonuclease in 225µL Exonuclease buffer, and gently tap to mix. Let sit at room temperature for 15 minutes. 15. Remove ethanol and place tube upside down on a paper towel for a few minutes. Then, lay tube on its side for 5 minutes. After this, use a sterile Q-tip to remove any remaining ethanol on the sides of the tube. EXONUCLEASE DIGESTION OF CHROMOSOMAL DNA: 17. Add 200µL ATP-dependent exonuclease and 300µL ATP solution to dissolved DNA. Mix gently by carefully rocking the tube, and incubate at 37°C for 1 hr. (After this incubation if there is still white debris, do a 5 minute spin >15,000xg, and carefully remove the supernatant into a new container. If there is a lot of debris left, it will clog the columns). 18. Equilibrate Qiagen-Tip 500 by applying 10mls buffer QBT to column and empty by gravity flow. 19. Add 10mls buffer QS to DNA sample and apply the whole sample to the column by pouring it on; let it enter by gravity flow. 20. Wash the tip twice with 30mls of buffer QC. 21. Elute DNA three times with 5mls of buffer QF, prewarmed to 65°C. 22. Precipitate DNA with 10.5mls (0.7vol) room temp isopropanol. Mix well and immediately cntrigue >15,000g for 30 minutes, 4°C. Carefully decant supernatant. 23. Wash pellet with 5 mls room temperature 70% ethanol, and centrifuge >15,000g 15 minutes, 4°C. 24. Air dry pellet until there is no more ethanol on the sides of the tube, but do not overdry! Note, at this point the pellet is usually so clean you cannot see it. Drying takes ~15 minutes. I usually get impatient and then use a sterile Q-tip to get rid of the excess alcohol without disturbing the pellet.. 25. Add 600µL of T10E1 (10Mm Tris, 1Mm EDTA) and bathe pellet overnight at 4°C. Then next day, incubate at 65°C to ensure DNA is fully resuspended. Spin tube very briefly to collect liquid at bottom, and transfer to an eppendorf tube. |