Do not over-amplify the mucoid library, as this will inevitably cause distortion of the original genome. Faster-growing clones will be over-presented, unstable clones will undergo rearrangements, and slower-growing clones may disappear from the library altogether. 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 in liquid medium)
Principle
Do not over-amplify the mucoid library, as this will inevitably cause distortion of the original genome. Faster-growing clones will be over-presented, unstable clones will undergo rearrangements, and slower-growing clones may disappear from the library altogether.
Materials and Instruments
λ Phage packaging reaction E. coli inoculation strain Move I. Materials For more product details, please visit Aladdin Scientific website.
Glycerol
Agar plate for kanamycin TB medium Sorvall GSA Turn head or equivalent Cryotubes
1. Buffers and solutions
Glycerol
2. Culture media
Agar plate containing 25 μg/ml kanamycin
TB medium
TB medium with 25 μg/ml kanamycin
3. Centrifuges and rotors
Sorvall GSA head or equivalent
4. Specialized equipment
Cryotubes
5. Vectors 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 that can produce 30,000~50,000 transformed colonies.
2. Take a number of sterile test tubes and add 0.2 ml of inoculated bacteria to each tube, then add the Packaging Reaction Solution and bring it up to the volume determined in step 1.
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.
Amplification was performed in TB medium containing 25 μg/ml kanamycin.
5. 0.25 ml of each infected cell was inoculated into 100 ml of TB medium containing 25 μg/ml kanamycin and placed in 250 ml culture flasks.
6. Shake the culture vigorously at 37℃ to make the cell density to OD600=0.5~1.0.
7. The cultures were combined, centrifuged at 5000 g (5500 r/min, Sorvall GSA turntable) at 4℃ for 10 min, recovered, and resuspended with 1/10 of the original volume of TB.
8. Add sterile glycerol to the cell suspension to a final concentration of 15% (V/V). Invert the sealed tube several times to mix the suspension well. Dispense the bacterial suspension in 0.5-1.0 ml volume into several sterile tubes, seal the tubes and store at -70°C.
9. For screening, rapidly thaw a small portion of frozen cells at 37°C and spread them onto numbered filter membranes at a density of 30,000 to 50,000 bacteria per membrane. The colonies are lysed on photocopied membranes, which are then hybridized to the labeled probes. 