Isolation of BAC DNA from bulk cultures
Isolation of BAC DNA from bulk cultures
The amount of DNA required for fine analysis of recombinant BAC, including detailed restriction mapping, DNA sequencing or subcloning, is greater than can be provided by small-volume preparations. This procedure is suitable for large-scale cultures of recombinant BAC. 500 ml of BAC transformants yields an average of 20-25 μg of BAC DNA, and the DNA product can be further purified by column chromatography. This protocol is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang, Peitang et al.
Operation method
Isolation of BAC DNA from bulk cultures
Principle
The amount of DNA required for fine analysis of recombinant BAC, including detailed restriction mapping, DNA sequencing or subcloning, is greater than can be provided by small-volume preparations. This protocol is suitable for large-scale cultures of recombinant BAC. the average yield of 500 ml of BAC transformants is 20-25 μg of BAC DNA. the DNA product can be further purified by column chromatography. the product can be used as an analyte for the analysis of BAC recombinants.
Materials and Instruments
Lysozyme Restriction Endonuclease DNA Standard Reference Chromatography Resin Escherichia coli Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol Isopropanol DNA extract Alkaline lysate STE solution TE
Pulsed Field Gel Electrophoresis Instrument LB medium Sorvall GSA Turn Head or equivalent
1. Buffers and solutions
Ethanol
Isopropyl alcohol
Phenol/chloroform (1:1, V/V)
DNA Extraction Solution
Alkaline lysate I, pre-cooled
Alkaline lysis solution Ⅱ
Alkaline lysate Ⅲ, pre-cooled
STE solution, pre-cooled
TE (pH 8.0)
2. Enzyme and buffer
Lysozyme
Restriction endonucleases
3. Nucleotides and oligonucleotides
DNA standard reference for pulsed-field gel electrophoresis
4. gels
Pulsed Field Gel Electrophoresis Instrument
5. Culture media
LB medium containing 12.5 μg/ml chloramphenicol
6. centrifuge and rotor
Sorvall GSA head or equivalent with Oakridge tubes (Nalgene)
7. Specialized equipment
Chromatography Resin
8. Vectors and strains
BAC-transformed E. coli
II. METHODS
1. Inoculate 50 μl of BAC-transformed bacteria from overnight culture into 500 ml of LB medium containing 12.5 μg/ml chloramphenicol, and incubate at 37℃ with vigorous shaking (300 r/min) for 12-16 h until the cells are saturated.
2. Centrifuge the cells at 2500 g (Sorvall GSA turning head 3900 r/min) for 15 min at 4℃, collect the cells, discard the supernatant, and invert the centrifuge flask so that the last drop of supernatant can be drained.
3. Resuspend the bacterial precipitate with 100 ml of ice pre-cooled STE. Collect the bacteria by centrifugation as in step 2.
4. Lysate the bacteria with 24 ml of Alkaline Lysis Solution I containing DNase-free RNAase (100 μg/ml). Add lysozyme to a final concentration of 1 mg/ml.
5. Add 24 ml of freshly prepared Alkaline Lysate II. Tightly cap the centrifuge bottle, gently turn the bottle several times to mix the contents completely, and ice bath for 5 min.
6. Add 24 ml of ice pre-cooled Alkaline Lysate III, tighten the cap of the centrifuge bottle, gently vortex the bottle to mix the contents completely until there is no obvious separation of the two phases, and place on ice for 5 min.
7. Centrifuge the bacterial lysate at 15,000 g (Sorvall GSA turning head 9600 r/min) for 10 min at 4°C and transfer the supernatant to another polypropylene centrifuge bottle. Discard the precipitate in the centrifuge bottle.
8. Add equal volumes of phenol: chloroform. Gently turn the centrifuge bottle several times to mix the aqueous and organic phases. Separate the two phases by centrifugation at 3000 g (Sorvall GSA turntable 4300 r/min) for 15 min at room temperature.
9. Transfer the aqueous phase to a new centrifuge bottle with a wide-bore pipette tip, add an equal volume of isopropanol, turn the bottle several times and mix well.
10. Mark one side of the bottle and place the marked side away from the center of the rotor head. This marking will help to locate the nucleic acid precipitate next. Centrifuge at 15,000 g (Sorvall GSA turntable 9600 r/min) for 15 min at room temperature to recover the nucleic acid precipitate.
11. Carefully discard the supernatant and place the centrifuge bottle upside down on a piece of paper until the last drop of supernatant is drained. Rinse the precipitate and tube walls with 20 ml of 70% ethanol at room temperature. Absorb the ethanol and place the bottle upside down on a stack of paper towels for a few minutes at room temperature to allow the ethanol to evaporate.
12. Carefully dissolve the BAC DNA precipitate in 0.2 ml TE (pH 8.0). Flick the walls of the tube without oscillating on the vortexer to promote DNA dissolution. Measure DNA concentration by spectral absorption.
13. Digest the DNA with restriction endonuclease.
14. analyze digested BAC DNA by PFGE. electrophoresis should be performed using an appropriately sized DNA standard reference.