This method was reported by Hanahan and Meselson (1980, 1983) and is primarily used to treat bacteria transformed by plasmid cDNA libraries. A mixture of transformed bacteria or a copy of the amplified cDNA library is placed directly onto a detergent-free nitrocellulose or nylon membrane, and the replica membrane is prepared by membrane-to-membrane contact. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Screening experiments of large numbers of bacterial clones by hybridization methods
Principle
This method was reported by Hanahan and Meselson (1980, 1983) and is primarily used to treat bacteria transformed by plasmid cDNA libraries. A mixture of transformed bacteria or a copy of the amplified cDNA library is placed directly onto detergent-free nitrocellulose or nylon membranes, and replica membranes are prepared by membrane-to-membrane contact. Using this method, 2X104 clones per 150 mm plate or 1X104 clones per 90 mm plate can be replicated and screened for further hybridization.
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 Flat-tip forceps 18-gauge needle Cellulose nitrate membrane Syringe Whatman 3 MM round filter paper
1. Culture medium
(1) LB or SOB agar plates containing appropriate antimicrobials.
In this program, 2-3 days old plates are used for best results because they are more likely to absorb water from the inoculum.
(2) LB or SOB agar plates containing appropriate antimicrobials and 25% (V/V) glycerol.
(3) LB or SOB agar plates containing chloramphenicol.
The concentration of chloramphenicol storage solution should be 170-200 μg/ml.
2. Specialized equipment
Flat-tip tweezers (e.g. Millipore tweezers)
18-gauge needle
Nitrocellulose membrane (Millipore HATF, or equivalent) or nylon membrane, sterile and free of detergent contamination
Syringe (3 cc)
Whatman 3 MM round filter paper. (Prepare a stack of Whatman 3 MM filter papers (one for each nitrocellulose or nylon membrane, plus a few extras) and cut them slightly larger than the membranes. Some manufacturers sell pre-made round filter papers. Wrap the paper in lead foil and autoclave for 10 minutes at 15 psi (1.05 kg/cm2 ).
3. Carriers and Strains
Recombinant plasmid-transformed E. coli is used as a culture.
or
amplified cDNA libraries, grown as cultures.
II. Methods
1. 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 with aluminum foil, and autoclave in a 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.
2. Using flat-tipped forceps, place a sterilized membrane, labeled side down, on an LB or SOB agar plate containing the appropriate antimicrobial agent that has been prepared for 2-3 days. After the membrane is completely wetted, remove it from the plate, turn it over labeled side up, and place it on the surface of the agar plate.
3. Place a small volume of the bacterial solution in the center of the filter membrane on the surface of the agar plate, use a sterile glass rod to disperse the bacterial solution, leaving a sterile zone 2~3 mm wide around the filter membrane.
The large membrane (137 mm diameter) provides 0.5 ml of bacterial suspension containing 2X104 viable bacteria, while the small membrane (82 mm diameter) provides 0.2 ml of bacterial suspension containing ~104 viable bacteria.
4. Place the agar plate with the lid half open (not inverted) in a fume hood for a few minutes to allow water to evaporate from the inoculum, close the lid, and incubate the plate upside down at 37°C until small colonies of 0.1-0.2 mm in diameter appear (about 8-10 h).
5. If required, a back-up membrane can be prepared at this point according to step 6, otherwise the colonies can be frozen at -20°C according to the following procedure:
(1) Transfer the filter membrane colony side up onto the surface of an LB or SOB agar plate containing the appropriate antimicrobial and 25% glycerol.
(2) Incubate for 2 h at 37°C.
If screening cDNA libraries, it is best to freeze the plates. Freezing reduces fungal contamination and allows the plates to be stored for several months. Main filter membranes on LB or SOB plates containing antimicrobials but no glycerol can be stored at 4°C for up to 2 weeks.
(3) Seal the agar plates with Parafilm and store upside down in a closed plastic bag at -20 °C.
Allow the plates to thaw at room temperature (still in the inverted position) and make a backup.
6. Place the colony side of the primary membrane (nitrocellulose or nylon) on sterile Whatman 3 MM filter paper.
7. Mark a piece of wet nitrocellulose or nylon membrane and place it on top of the primary membrane to prevent air bubbles from forming between the two membranes.
It is best to curl the second membrane slightly so that the two membranes are first in contact in the center. Once the two membranes are in contact, do not move them away from each other. Try not to overlap the two membranes completely so that they can be separated later.
8. Cover the membrane with another round piece of 3 MM filter paper and place a petri dish on top of the paper, pressing down firmly on the dish with the palm of your hand to transfer bacteria from the primary membrane to the filter membrane.
9. Remove the petri dish and the top layer of 3 MM filter paper, and mark the orientation of the double layer of filter membrane by making a number of blanks with an 18-gauge needle attached to a syringe.
Make sure the needle pierces both membranes.
10. Separate the two membranes and place the backup membrane on a fresh LB (or SOB) agar plate with appropriate antimicrobials.
At this point, a second backup can be prepared step-by-step, labeling the second backup membrane with the existing holes in the primary membrane.
11. Place the other backup membrane and the primary membrane on fresh LB (or SOB) agar plates containing appropriate antimicrobials and incubate at 37°C until colonies develop (4-6 h).
12. At this stage, when bacterial growth is still rapid, the membrane can be transferred to chloramphenicol-containing (170-200 μg/ml) (or SOB) agar plates and incubated at 37℃ for 12 h. The membrane is then transferred to a fresh LB (or SOB) agar plate containing the appropriate antimicrobial agent.
13. Transfer the primary membrane to fresh LB (or SOB) agar plates containing appropriate antimicrobials and 25% glycerol and freeze as in step 5.
14. Lyses colonies attached to the back-up membrane and immobilizes the released DNA to a nitrocellulose or nylon membrane. Hybridize.