Hybridization of bacterial DNA on filter membranes
Hybridization of bacterial DNA on filter membranes
This protocol describes how to hybridize radiolabeled probes to DNA of transforming bacteria immobilized on filter membranes, and methods for recovering clones from agar plates that hybridize specifically to the probes for probes with an average length greater than 100 nucleotides. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Hybridization of bacterial DNA on filter membranes
Principle
This protocol describes how to hybridize radiolabeled probes to DNA of transforming bacteria immobilized on filter membranes and methods for recovering clones from agar plates in culture that hybridize specifically to the probes, which are applicable to probes with an average length greater than 100 nucleotides.
Materials and Instruments
Filter membrane with immobilized transformed clone DNA Oligonucleotide probe Move I. Materials For more product details, please visit Aladdin Scientific website.
Formaldehyde Pre-hybridization Hybridization solution BLOTTO pre-wash Elution solution
LB, TY or Terrific broths
1. Buffers and solvents
Formaldehyde, prehybridization/hybridization solution, 1X BLOTTO, prewash solution (6X SSC or 6X SSPE), eluent 1 (2X SSC, 0.1% (m/V) SDS), eluent 2 (1X SSC, 0.1% (m/V) SDS), eluent 3 (0.1X SSC, 0.1% (m/V) SDS).
2. culture medium
LB, TY or Terrific broth containing appropriate antimicrobials.
3. Nucleic acids and oligonucleotides
Filter membranes with immobilized transformed cloned DNA.
4. probes
32P-labeled double-stranded DNA probes or synthetic oligonucleotide probes.
5. Specialized equipment
(1) Water bath
(2) Glass baking dish (15 cm X 7.5 cm X 5 cm ) or other hybridization container
(3) Incubator with adjustable temperature up to 42°C (e.g. prehybridization in formaldehyde), 50°C and 68°C
(4) Radioactive ink
(5) Hydrocolloid strips
(6) Whatman 3 MM filter paper or equivalent
(7) Wooden toothpicks or inoculation rings
II.
Pre-elution and pre-hybridization of filter membranes
1. Float the baked or crosslinked membranes on the surface of 2X SSC liquid in a tray until the membranes are completely wetted from the bottom, then soak for 5 min.
Because some batches of nitrocellulose membranes can swell and cross-link during hybridization and drying, it can be difficult to align membranes or agar plates with the directional markings on the autoradiograph. This problem can be partially alleviated by autoclaving [10 psi (0.70 kg/cm2 ) for 10 min] the dry membrane between wet 3 MM filter paper before use. This problem does not occur with nylon membranes.
2. Transfer the membranes to a glass baking dish containing at least 200 ml of pre-elution solution, stack the membranes in the liquid, seal the baking dish with plastic wrap, place on a rotating platform in the incubator, and hold at 50°C for 30 min.
Pre-elution removes bacterial clone residues and significantly reduces hybridization background, especially when screening for very dense clones.
Here and in all subsequent steps, the filter membranes should be shaken slowly to prevent sticking to each other. Do not allow the filter membrane to dry during all processes such as pre-washing, pre-hybridization or hybridization.
3. Scrape the bacterial debris off the surface of the membrane with Kimwipes soaked in pre-elution solution; scraping off the bacterial debris will not affect the density and clarity of the positive hybridization signal.
4. Transfer the membrane to a glass baking dish containing 150 ml of prehybridization solution and slowly shake the membrane at the appropriate temperature (e.g., 68°C for hybridization in aqueous solution; 42°C for hybridization in 50% formaldehyde) for 1 to 2 h or longer.
The filter membrane should be completely covered by the prehybridization solution. During prehybridization, sites on the nitrocellulose membrane that are nonspecifically bound by single- or double-stranded DNA should be closed by proteins in the BLOTTO solution. Shaking allows the membrane to be continuously bathed and covered by the prehybridization solution.
Probe Denaturation and Hybridization Filter Membranes
5. Denature the 32P-labeled double-stranded DNA probe by heating at 100°C for 5 min and immediately place in an ice bath.
The probe can also be denatured by adding 0.1 mol/L NaOH by volume. After 5 min at room temperature, transfer the probe to an ice bath and add 0.05 v/v of 1 mol/L Tris-HCl (pH 7.2) and 0.1 v/v of 3 mol/L HCl. Keep the probe in the ice bath until use.
Single-stranded probes do not require denaturation. 
6. Add the probe to the membrane-covered prehybridization solution and incubate at the appropriate temperature until the C0t1/2 reaches 1 to 3. During hybridization, the container should be sealed to prevent loss of liquid by evaporation.
7. After hybridization, pour off the hybridization solution and quickly immerse the membrane in a large volume (300-500 ml) of Wash Solution 1, shaking the membrane slowly and turning it over at least once. 5 min later, transfer the membrane to a container of new Wash Solution and continue to shake it slowly. Repeat the membrane wash more than twice.
8. Wash the membrane twice with 300-500 ml of Wash Solution 2 within 0.5-1.5 h at 68℃.
9. Place the membrane on 3 mm filter paper and dry at room temperature. Place a strip of water-soluble adhesive underneath the membrane, and then place the membrane on a clean, dry and flat piece of 3 mm filter paper and press the membrane firmly to make it adhere to the filter paper.
10. Mark several asymmetrical locations on the 3 mm filter paper with radioactive ink or chemiluminescent reagent. Cover the membrane and the marked paper with plastic wrap, adhere the wrap to the back of the paper with an adhesive strip, and spread the wrap over the membrane to remove any wrinkles.
These marks can be used to calibrate the self-revealing film against the filter paper.
Analysis of hybridization signals and identification of positive clones
11. Analyze filter membranes by acid photographic imaging or by exposure to X-ray film (Kodak XAR-2, XAR-5, or other equivalents) at -70°C for 12-16 h and sensitization screen.
12. X-rays are aligned with the membranes using marks left in radioactive ink, and non-radioactive fiber-tipped pencils are used to make marks on the numbered membranes in a non-black color that correspond to the location of the asymmetric points on the X-rays.
13. Place a piece of transparent paper over the x-ray film and mark on the paper the location of the positive hybridization signal and also the location of the asymmetry point (in a different color). Remove the transparency from the X-ray film and identify the positive clone by the correspondence between the markings on the transparency and the markings on the agar plate.
14. Transfer the positive clones to 1-2 ml of nutrient-rich medium (LB, YT or Terric broth) containing appropriate antimicrobials using a sterile toothpick or inoculation loop.
15. After a period of incubation, plasmid DNA can be extracted from the culture by one of the micro-preparation methods and further analyzed by restriction enzyme digestion or PCR.