Bacterial clones on the main agar plate and on a nitrocellulose filter membrane or nylon membrane covering the surface of another agar plate can be localized, and after a period of time, clones already grown on the filter membrane can be lysed in situ for hybridization. In the meantime, the master agar plate can be stored at 4°C until the results of the screening are available. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Screening experiments of small quantities of bacterial clones by hybridization method
Principle
Bacterial clones on the main agar plate and on a nitrocellulose filter membrane or nylon membrane covering the surface of another agar plate can be localized, and after a period of time, clones already grown on the filter membrane can be lysed in situ for hybridization. In the meantime, the master agar plate can be stored at 4°C until the results of the screening are available.
Materials and Instruments
E.coli Move I. Materials For more product details, please visit Aladdin Scientific website.
LB or SOB agar plates
Nitrocellulose membrane Syringe Needle Waterproof black ink Wooden toothpick or inoculation loop
1. Culture medium
LB or SOB agar plates containing appropriate antimicrobials.
LB or SOB agar plates containing chloramphenicol.
2. Specialized equipment
(1) Cellulose nitrate membrane (Millipore HAWP, or equivalent) or nylon membrane.
The membrane need not be free of detergent contamination or sterile.
(2) Syringe (3cc ), needle (18-gauge) and waterproof black ink (India ink)
The above materials are used to mark the orientation of the filter membrane on the main agar plate.
(3) Wooden toothpick or inoculation ring.
3. Carriers and strains
E.coli for recombinant plasmid transformation.
E.coli for non-recombinant plasmid transformation (e.g. pUC, as a negative control).
Methods
1. Nitrocellulose or nylon membranes were spread on agar plates (experimental plates) containing selective antimicrobials.
Gloves should be worn when handling the membrane, as oil from fingers can interfere with membrane wetting and impede DNA transfer.
2. Draw a numbered grid (1 cm2 ) on a piece of graph paper, mark the number on the bottom of each agar plate, place the agar plate on the grid, and mark the 6 o'clock position on the edge of the agar plate.
This marking will align the motherboard with the orientation of the squares.
3. Use a sterile toothpick or inoculating loop to transfer colonies one by one to the filter membrane of the plate and to the main plate containing the selective antimicrobial (no filter membrane). Mark the colonies as short 2-3 mm lines or dots according to the grid below the plate, with each colony in the same position in both plates.
One 90 mm plate can contain 100 clones.
4. Finally, one clone containing a non-recombinant plasmid (e.g., pUC) is drawn on the filter membrane and one on the main agar plate.
Negative controls are necessary to distinguish the empty plasmid from the recombinant plasmid with a radioactive probe. DNA fragments extracted from restriction digests and agarose electrophoresis of recombinant plasmids are often contaminated with plasmid DNA sequences, which can be problematic when contaminated DNA fragments are used as hybridization probes for Grunstein-Hogness screening of transformant clones.
5. Invert the agar plate and incubate at 37°C until the width of the bacterial line is calibrated to 0.5-1.0 mm (usually 6-16 h).
At this stage, if the bacteria are still growing rapidly, the membranes should be transferred to chloramphenicol-containing agar plates and further incubated at 37°C for 12 h (Hanahan and Meselson 1980, 1983). This amplification process is only necessary if the copy number of the recombinant plasmid is expected to be low (e.g., if a large exogenous DNA fragment is inserted) or if a highly condensed oligonucleotide is used as a probe. Cloned DNA fragments are usually easily detected by hybridization and do not require prior amplification of the recombinant plasmid. Amplification is only effective for plasmids that replicate in a loose manner.
6. Mark the filter membrane at three or more asymmetric locations and puncture the membrane into the agar of the plate with an 18-gauge needle attached to a syringe filled with waterproof ink. Mark similarly on the main plate in close proximity.
In practice, it is possible to puncture holes between the filter membrane and the underlying agar with an empty 18-gauge needle without using ink (which can stain), and after hybridization, the holes in the filter flags can be checked against the traces on the agar by backlighting from the light box.
Many researchers prefer the method of using scissors to cut notches at various locations around the filter membrane to determine orientation. The notched and labeled filter membrane is placed on the agar surface, and the location of the serrated edges of the filter membrane are later marked on the back of the culture plate. In this way, the shape and position of the serrations can be used to determine the orientation on the culture plate.
7. Seal the plates with Parafilm, turn upside down and store at 4°C until the results of the hybridization experiments are available.
8. Lyses the bacteria attached to the membrane and binds the released DNA to a nitrocellulose or nylon membrane. Hybridization is performed.