I use QIAGEN Plasmid Midi kit. 100ml culture could give about 30-50ug. I
tried Qiagen's large construct kit.But in my hand, the midi kit is much much
better than the large construct kit, and also takes much less time.The
following is the protocol I got from Qiagen.
User-Developed Protocol:
Isolation of BAC DNA using the QIAGEN® Plasmid Midi Kit
This procedure has been adapted by customers from the QIAGEN® Plasmid
Midi Kit Protocol.It has not been thoroughly tested and optimized by QIAGEN.
This procedure has been used successfully for isolation of 150–250 kb BAC
DNA from a mouse-BAC library cloned in pBeloBAC11 from Escherichia coli
strain HB101/r. The yield of BAC DNA from 100 ml culture was typically 20–
40 μg.
Please be sure to read the QIAGEN Plasmid Purification Handbook and the
detailed QIAGEN Plasmid Midi Kit Protocol carefully before beginning this
procedure.
Procedure
1. Pick a single BAC colony and inoculate a starter culture of 5 ml LB
medium containing the appropriate antibiotic.
2. Inoculate 0.5 ml pre-culture into 100 ml selective LB medium. Grow at 37
°C for 14 hours with vigorous shaking (~250 rpm).
3. Divide the cells into two 50 ml tubes, and harvest the cells by
centrifugation at 4500 x g for 20 min.
4. Resuspend each bacterial pellet in 10 ml Buffer P1.
Ensure that RNase A (100 μg/ml) has been added to Buffer P1.
5. Add 10 ml Buffer P2 to each tube. Mix thoroughly and gently by inverting
4–6 times,and incubate at room temperature for 5 min.
Check Buffer P2 before use for SDS precipitation due to low storage
temperatures. If necessary, dissolve the SDS by warming to 37°C.
6. Add 10 ml chilled Buffer P3 to each tube. Immediately mix by gently
inverting 4–6 times, and incubate on ice for 15 min.
7. Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant
containing plasmid DNA promptly.
8. Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C.
Remove supernatant containing plasmid DNA promptly.
9. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the
column to empty by gravity flow.
10. Pool the two supernatants from step 8. Apply the sample to the QIAGEN-
tip and allow it to enter the resin by gravity flow.
11. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC.
12. Elute DNA with 5 x 1 ml Buffer QF, prewarmed to 65°C.
Prewarming the elution buffer may help to increase yields. Eluting in 5
aliquots of 1 ml instead of 1 aliquot of 5 ml prevents cooling of the
elution buffer.
13. Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the
eluted DNA.
Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. Carefully
decant the supernatant.
14. Wash the DNA pellet with 2 ml of room-temperature 70% ethanol and
centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant
without disturbing the pellet.
15. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable
volume of buffer (e.g., TE, pH 8.0, or 10 mM Tris·Cl, pH 8.5).
