Applying this amplification method, the library is not prone to distortion, due to the fact that mixed colonies containing different recombinant mucilages do not compete in growth at any step. However, the amplification process is lengthy and is sometimes lost due to the fact that the colonies no longer grow after the primary filter membrane is preserved. This section also provides an alternative method of amplification on TB plates. This alternative amplification scheme makes it easy to lose those poorly growing mucoid clones. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" Translated by Huang Peitang et al.
Operation method
Amplification and storage of mucoid libraries (amplification on filter membranes)
Principle
With this amplification method, the library is not susceptible to distortion due to the fact that mixed colonies containing different recombinant mucilages do not compete for growth at any step. However, the amplification process is lengthy and is sometimes lost due to the fact that the colonies no longer grow after the primary filter membrane is preserved. This section also provides an alternative method of amplification on TB plates. This alternative amplification scheme makes it easy to lose those poorly growing mucoid clones.
Materials and Instruments
λ Phage packaging reaction E. coli inoculated strains Move I. Materials For more product details, please visit Aladdin Scientific website.
Kanamycin TB Agar Plate TB Medium Non-detergent, sterile nitrocellulose or nylon filter membrane
1. Culture medium
Agar plates (150 mm) containing 25 μg/ml kanamycin TB.
TB medium
2. Specialized equipment
Degreaser-free, sterile nitrocellulose or nylon filter membrane (137 mm).
3. Carriers and strains
λ Phage packaging reaction
Suitable E. coli inoculation strains (e.g. XLl-Blue, ED8767, NM554, DH5αMCR)
II. Methods
Pre-culture
1. Calculate the packaging reaction volume of colonies that can produce 30,000~50,000 transformations.
2. Take a number of sterile test tubes and add 0.2 ml of inoculated bacteria to each tube, followed by the Packaging Reaction Solution and bring it up to the volume determined in Step 1.
3. Incubate the tubes at 37°C for 20 min.
4. Add 0.5 ml of TB to each tube and continue incubation for 45 min.
Amplification on filter membrane
5. Spread numbered sterile filter membranes onto several 150 mm TB agar plates containing 25 μg/ml kanamycin (the same number of membranes as in step 2).
6. Apply the contents of each tube to the surface of the filter membrane on the agar plate using a sterile applicator stick. After the inoculum has been absorbed by the membrane, transfer the plate to a 37°C incubator and leave for several hours to overnight (12-15 h).
7. Make a photocopy of each primary filter membrane and store at -70°C.
8. For library screening, melt the photofilm, lyse the clones on the filter, and then hybridize to the labeled probes. 
