Screening experiments of intermediate cell clones by hybridization method
Screening experiments of intermediate cell clones by hybridization method
This method is applicable to bacterial clones of any size, but small clones give the clearest results. Small clones produce sharp hybridization signals and are not diffused during the transfer of clones from the agar plate to the membrane. A 90 mm plate capable of containing 2500 clones is best for this operation. It is difficult to cover a 150 mm plate exactly with a larger membrane while ensuring that there are no air bubbles. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Screening experiments of intermediate cell clones by hybridization method
Principle
This method is applicable to bacterial clones of any size, but small clones give the clearest results. Small clones produce sharp hybridization signals and are not diffused during the transfer of clones from the agar plate to the membrane. A 90 mm plate capable of containing 2500 clones is best for this operation. It is difficult to cover a 150 mm plate exactly with a larger membrane while ensuring that there are no air bubbles.
Materials and Instruments
Recombinant plasmid transformed E.coli Move I. Materials For more product details, please visit Aladdin Scientific website.
LB or SOB agar plates Cellulose nitrate membranes Syringes Needles Waterproof black ink Whatman 3 MM round filter paper
1. Culture medium
(1) LB or SOB agar plates (90 mm) containing appropriate antimicrobials.
The best results are obtained by using 2-3 days old plates in this program because they are more likely to absorb water from the inoculum.
(2) LB or SOB agar plates containing chloramphenicol.
The concentration of chloramphenicol storage solution should be 170~200 μg/ml.
2. Specialized equipment
(1) Cellulose nitrate membrane (Millipore HATF, or equivalent) or nylon membrane, sterile and free of detergent contamination.
(2) Syringe (3 cc), needle (23-gauge) and waterproof black ink (India ink).
The above materials are used to mark the orientation of the filter membrane on the agar plate.
(3) Whatman 3 MM round filter paper.
Prepare a stack of Whatman 3MM filter paper (one stack for each nitrocellulose or nylon membrane, plus a few extras) in advance and cut it down to a size slightly larger than the membrane. Some manufacturers have pre-made round filter papers available.
3. Carriers and Strains
Recombinant plasmid transformed E. coli, used as culture.
II. Methods
1. Spread the transformed culture on 90 mm LB or SOB agar plates, and adjust the concentration of the liquid to allow the growth of 2500 colonies. After the colonies have grown to an average size of 1.5 mm, move the agar plate from the incubator to a cold room.
2. Mark the dry filter membrane with a soft pencil or ballpoint pen, moisten with water, sandwich the wet membrane between dry 3 mm filter paper, wrap loosely in aluminum foil, and autoclave the liquid-phase cycle [15 psi (1.05 kg/cm2 ) for 10 min].
Prepare enough membranes to make 1 or 2 backups from the starting plate. Sterilized sterile membranes are preferred for backups, but unsterilized membranes can be used if backups are no longer prepared from the master plate.
3. Lay dry, sterile, detergent-free nitrocellulose membrane marker side down on the surface of the LB or SOB agar plate in contact with the colonies (step 1 completed) until the membrane is completely wetted.
Be careful to prevent air bubbles from forming between the membrane and the agar, which can cause artifacts. It is best to curl the membrane slightly so that the center of the membrane contacts the agar first. Do not move the membrane after it has come into contact with the agar.
4. As soon as the membrane is in place, mark three or more asymmetrical locations with a 23-gauge needle attached to a syringe moistened with waterproof ink.
A 23-gauge needle or smaller ink dab is best (one dab per plate, three holes per plate).
In practice, it is possible to use an empty 18-gauge needle to perforate between the filter membrane and the lower layer of spheroplasts, avoiding the use of ink (which can get dirty). After hybridization, backlighting from the light box allows the holes in the filter membrane to be calibrated against the traces on the agar.
Many researchers prefer the method of using scissors to cut notches at different locations around the filter membrane to determine orientation. The notched and labeled membrane is placed on the agar surface and the location of the serrated edge of the membrane is marked on the back of the plate. In this way, the shape and position of the serrations can be used to determine the orientation on the culture plate.
5. Remove the membrane from the surface of the agar plate in one smooth, single motion by holding the edge of the membrane tightly with flat-nosed forceps.
Usually the clone is transferred from the agar to the membrane in one piece, but it will leave a dent in the surface of the medium, which will be filled with bacteria growth but will not expand when the plate is incubated at 37 °C.
6. Perform one of the following procedures, as appropriate:
(1) Lyses the colony on the membrane and immobilizes the released DNA on a nitrocellulose or nylon membrane. Perform hybridization.
(2) Lysing the colony and replacing the immobilized DNA.
(3) Place the colony side of the filter membrane upwards on the surface of a new LB (or SOB) agar plate containing appropriate antimicrobials and incubate for a few hours. After the colonies have grown to 2~3 mm, remove the filter membrane for lysis and hybridization.
The above operation is performed only when the number of clones on the plate is small or uneven when the clones are transferred to the filter membrane. However, this is not common.
(4) Place the filter membrane on an agar plate containing chloramphenicol (170~200 μg/ml) and incubate at 37℃ for a further 12 h for amplification, followed by lysis and hybridization.
This amplification process is only necessary when the copy number of the recombinant plasmid is expected to be low (e.g., when a large exogenous DNA fragment is inserted) or when oligonucleotides with a high degree of parsimony are used as probes. 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.
(5) Preparation of secondary backups with filter membranes:
① Place the colony side of the filter membrane up on the surface of a new LB or SOB agar plate containing the appropriate antimicrobial.
② Carefully place a piece of dry nitrocellulose membrane on top of the first membrane and label it as described in Step 4 above.
② Carefully place a dry nitrocellulose membrane on top of the first membrane and label it as described in step 4 above. ③ Incubate the "membrane sandwich" at 37℃ for several hours.
If necessary, incubate further on agar plates containing chloramphenicol to amplify the plasmid.
④ Perform lysis and hybridization, keeping the filter membrane sandwich in place during the lysis and neutralization steps, and removing it during the final elution (Ish-Horowicz and Burke. 1981).
7. The master plate was incubated at 37℃ for 5~7 h until the colonies grew again, sealed with Parafilm membrane, inverted and stored at 4℃.