Screening of unamplified mucoid libraries by hybridization (filter film photocopying)
Screening of unamplified mucoid libraries by hybridization (filter film photocopying)
Thick bacterial colonies transformed by mucilage can be screened by hybridization, a method derived from the large-scale screening of plasmid-transformed colonies (Hanahan and Meselon 1980). The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Screening of unamplified mucoid libraries by hybridization (filter film photocopying)
Principle
Thick bacterial colonies transformed by mucilage can be screened by hybridization, a method derived from the large-scale screening of plasmid-transformed colonies (Hanahan and Meselon 1980). Move I. Materials For more product details, please visit Aladdin Scientific website.
1. Culture medium
TB agar plates (150 mm ) containing 25 μg/ml kanamycin.
TB medium
2. Specialized equipment
Thick glass plate
Hypodermic needles (17 gauge)
Sterilized nitrocellulose or nylon membrane (137 mm)
Sterilized Whatman No. 1 filter paper
3. Carriers and strains
λ Phage Packaging Reaction
E. coli strains suitable for plate inoculation (e.g. XL1-BLUE, ED8767, NM554, DH5αMCR).
II. Methods
Mucoid DNA that has been packaged into λ phage particles is transformed into E. coli
1. Calculate the volume of the packaging reaction that produces 30,000~50,000 transformed colonies.
2. Prepare several sterilized test tubes with the above volume of packaging reaction product and 0.2 ml of inoculum bacteria.
3. incubate the tubes at 37℃ for 20 min.
4. Add 0.5 ml of TB to each tube and continue incubation for 45 min.
5. Spread the sterile filter membrane onto a 150 mm TB agar plate containing 25 μg/ml kanamycin (the number of plates required is equal to the number of tubes in step 2).
6. Using a sterilized applicator stick, spread the mixture from each tube evenly onto the filter membrane of the agar plate. When the inoculum has been absorbed by the membrane, place the plate in a 37°C incubator for several hours or overnight (12-15 h).
7. Spread a 137 mm numbered sterile filter membrane onto a fresh TB agar plate containing 25 μg/ml.
8. Place a piece of sterile Whatman No. 1 filter paper on a thick sterile glass plate.
9. Transfer the photocopied filter membrane from the fresh TB agar plate (step 7 ) to the Whatman No. 1 filter paper using sterile flat-tipped tweezers.
10. Remove the main filter membrane with the transformed colonies from the TB agar plate (step 6) with tweezers and place the colonies face down exactly on the numbered photocopying filter membrane located on the Whatman No. 1 filter paper. Cover both filter membranes with another piece of sterile Whatman No. 1 filter paper.
11. Press the two membranes tightly together with a second sterile thick glass plate.
12. Remove the top plate of thick glass and the Whatman No. 1 filter paper and use a 17-gauge hypodermic needle to make a series of asymmetric holes (5 or so is sufficient) along the edge of each pair of nitrocellulose or nylon membranes to position the two membranes relative to each other.
Photocopying Filter Membrane Growth
13. Peel off the two membranes (nitrocellulose or nylon) and quickly resurface them on their respective TB agar plates (containing 25 μg/ml kanamycin).
14. Warm the primary and photocopy membranes at 37°C for several hours until the bacterial colonies are 0.5-1.0 mm in diameter.
15. Seal the master plate with a Parafilm membrane and store inverted at 4℃.
16. Lyses the colonies on the photocopied membrane and hybridizes the membrane with a radiolabeled probe.
The colorblot filter membrane can be used to replicate the library again, or stored at -70°C. 
