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| This protocol describes how to subclone inserts from a BAC or P1 clone into a M13 vector to generate a M13 library. Bacterial artificial chromosome (BAC) libraries are based on the naturally occurring F-factor plasmid found in the bacterium E. coli. With this system it is possible to maintain inserts of 100 kb to 300 kb in size as a single copy. The bacteriophage P1 system is another means of cloning large fragments of DNA that also maintains the clones as single copies in an E. coli host. Inserts from 70 kb to 100 kb can be cloned. Once a BAC or P1 clone has been identified as containing sequence of interest, it can be subcloned to construct a M13 library of smaller insert size for further screening and analysis. |
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1. Extract and purify DNA from a bacterial artificial chromosome (BAC) library clone or a bacteriophage P1 clone using a Plasmid Midi kit (Qiagen) or a BAC/P1 purification method of choice.
2. Add 10X Mung Bean Nuclease Buffer to approximately 5 μg of DNA and ddH2O to give a final concentration of 1X. The final volume should not exceed 60 μl.
2. Set a sonicator to setting 8 and continuous operation. Sonicate the DNA for 8 sec.
3. Centrifuge the tubes briefly in a microcentrifuge to collect the contents and add 10 Units of Mung Bean Nuclease per μg of DNA.
4. Mix gently and incubate for 30 min at 30°C.
5. After the incubation, add 6 μl of 6X DNA Loading Dye and load the DNA onto a 0.8% Low-Melt Agarose Gel in 1X TAE Buffer. Include the appropriate DNA size markers (see Scientific Reference Pages).
6. Electrophoresis the gel overnight at 23V (see Protocol on Agarose Gel Electrophoresis).
7. Soak the gel in Ethidium Bromide/1X TAE for 30 min and observe the gel under long-wave UV light.
8. Cut out the bands corresponding to the desired fragment size with a new razor blade.
9. Place the gel cutouts across the comb in a gel box and pour a 0.8% Low-Melt Agarose Gel around the gel cutouts.
10. Electrophoresis at 40 to 50V for 4 to 5 hr (see Hint #1).
11. Soak the gel in Ethidium Bromide/1X TAE for 30 min and observe the gel under long-wave UV light.
12. Cut out the bands corresponding to the desired fragment size and place each in a separate microcentrifuge tube.
13. Heat the tubes to 70°C for 10 min (or until the Agarose is completely melted).
14 Add 500 μl of Phenol per tube, mix by inversion, and centrifuge for 5 min at maximum speed in a microcentrifuge to separate the phases (CAUTION! see Hint #2).
15. Transfer the upper, aqueous phase to a new microcentrifuge tube, add another 500 μl of Phenol/tube, mix by inversion, and centrifuge for 5 min at maximum speed in a microcentrifuge to separate the phases.
16. Transfer the upper, aqueous phase to a new microcentrifuge tube and add 500 μl of Isopropanol per tube.
17. Incubate at -70°C from 30 min to overnight to precipitate the DNA.
18. Centrifuge at maximum speed for 20 min in a microcentrifuge.
19. Pour off the supernatant and add 1 ml of 70% Ethanol per tube to wash the DNA pellets.
20. Mix by inversion and centrifuge at the maximum speed for 5 min in a microcentrifuge.
21. Pour off the supernatant and invert the tubes over a paper towel to dry for approximately 15 min.
22. Resuspend the DNA pellet in 15 μl of TE Buffer. Combine the contents of any duplicate tubes into one tube.
23. Ligate the fragments into a vector by combining:
100 ng of DNA
100 ng of M13 vector (or suitable vector)
1 μl of 10X DNA Ligase Buffer
400 Units of DNA Ligase
Bring the reaction volume to 10 μl with ddH2O
Incubate at 16°C for 4 to 12 hours (determine empirically)
24. Transform the M13 vector (or suitable vector) using ultracompetent F' bacterial cells (see Protocol on Bacterial Transformation) (see Hint #3).
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| 70% (v/v) Ethanol |
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| Phenol |
| Store at 4°C in a dark glass bottle
Tris-equilibrated Phenol (CAUTION! see Hint #2)
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| 0.8% Low-Melt Agarose Gel |
| 1X TAE
Microwave to dissolve the Agarose
0.8% (w/v) Low-melt Agarose (SeaPlaque)
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| DNA Loading Dye (6X) |
| 0.25% (w/v) Bromophenol Blue
0.25% (w/v) Xylene Cyanol
30% (v/v) Glycerol
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| Ethidium Bromide/1X TAE |
| 0.5 μg/ml Ethidium Bromide (CAUTION! see Hint #2)
Prepare in 1X TAE
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| TAE Buffer (10X) |
| 37.2 g/liter EDTA, Disodium
57.1 ml/liter Glacial Acetic Acid
Adjust pH to 8.5
242 g/liter Tris Base
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| Mung Bean Nuclease Buffer (10X) |
| 500 mM NaCl
10 mM Zinc Chloride
50% (v/v) Glycerol
300 mM Sodium Acetate, pH 5.0
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| DNA Ligase Buffer (10X) |
| 50 mM DTT
200 mM Tris-Cl, pH 7.6
1 mM ATP
Or use the buffer supplied by a manufacturer of DNA Ligase
50 mM MgCl2
500 μg/ml BSA, Fraction V
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| TE buffer |
| 10 mM Tris
pH 8.0
0.1 mM EDTA
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EDTA, Disodium Salt
Ethanol
Magnesium Chloride
Phenol
Zinc Chloride
Bovine Serum Albumin (BSA)
Glycerol
Agarose, Low-Melt
Acetic Acid
DTT
Tris
DNA Ligase
Ethidium Bromide
Mung Bean Nuclease
Sodium Chloride
Glacial Acetic Acid
ATP
Isopropanol
Xylene Cyanol
Bromophenol Blue
Sodium Acetate
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1. Second electrophoresis times and voltage can be adjusted to optimize separation and recovery of different sized fragments. Determine this empirically.
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
3. The F' episome is a self-replicating plasmid that contains a defective lac operon.
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