Isolation of BAC DNA from small cultures
Isolation of BAC DNA from small cultures
Small amounts of BAC DNA were prepared from 5 ml of BAC-transformed cell cultures.The DNA was prepared by alkaline lysis.The yield of BAC DNA can be as high as 0.1~0.4 μg, which is sufficient for restriction enzyme digestion analysis, PCR or Southern blotting. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Isolation of BAC DNA from small cultures
Principle
Small amounts of BAC DNA were prepared from 5 ml of BAC-transformed cell cultures.The DNA was prepared by alkaline lysis.The yield of BAC DNA could be as high as 0.1-0.4 μg, which is sufficient for restriction enzyme digestion analysis, PCR or Southern blotting.
Materials and Instruments
Restriction endonuclease E. 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 DNA standard reference Sorvall SS-34 head or equivalent Chromatography resin
1. Buffers and solutions
Ethanol
Isopropyl alcohol
DNA extract
Alkaline lysis solution Ⅰ, pre-cooled
Alkaline lysis solution Ⅱ
Alkaline lysate Ⅲ, pre-cooled
STE solution, pre-cooled
TE ( pH 8.0)
2. enzymes and buffers
restriction endonuclease
3. gels
Pulsed-field gel electrophoresis apparatus
4. Culture medium
LB medium with 12.5 μg/ml chloramphenicol
5. Nucleotides and oligonucleotides
DNA Reference Standards for Pulsed Field Gel Electrophoresis
6. Centrifuges and Rotors
Sorvall SS-34 Turn Head or Equivalent
7. Specialized equipment
Chromatography Resin
8. Vectors and strains
BAC-transformed E. coli
II. METHODS
1. 5 ml of LB medium containing 12.5 μg/ml chloramphenicol was used to culture BAC-transformed E. coli, and the culture was shaken vigorously at 37℃ overnight.
2. Collect the bacteria by centrifugation at 2000 g (Sorvall SS-34 turn head, 4100 r/min) for 5 min at 4°C. Carefully discard the culture medium. Carefully discard the culture medium and remove the residue with a pipette.
3. Add 5 ml of pre-cooled STE to each centrifuge tube and blow up the bacterial precipitate with a pipette. Collect the bacteria by centrifugation as in step 2.
4. Resuspend the cells in 200 μl of ice-cold Alkaline Lysate I. Transfer the cells to a pre-cooled microfuge. Transfer cells to pre-cooled microcentrifuge tubes on ice.
5. Add 400 μl of freshly prepared Alkaline Lysate II to the tube. Gently turn the tightly capped centrifuge tube over several times. Place the tube on ice.
6. Add 300 μl of ice-cold Alkaline Lysate III to the tube and gently turn the tightly capped tube over several times. Place the tube on ice for 5 min.
7. Centrifuge in a microcentrifuge at maximum speed for 5 min at 4°C to remove the precipitate of cellular debris. Transfer the supernatant to a new microcentrifuge tube. Add 900 μl isopropanol at room temperature, gently turn the tube several times and mix well.
8. Immediately centrifuge in a microcentrifuge at maximum speed for 5 min at room temperature to precipitate the nucleic acids. Discard the supernatant and carefully rinse the precipitate with 1 ml of 70% ethanol. Centrifuge for 2 min at room temperature and aspirate the ethanol. Dry the precipitate at room temperature for 5-10 min and dissolve the wet precipitate in 50 μl TE (pH 8.0).
9. Digest the BAC DNA with restriction endonuclease.
10. Analyze the digested BAC DNA by PFGE, using a DNA standard reference of appropriate size for electrophoresis.