The library should be amplified as soon as possible once it is packaged; it can greatly increase the copy number of the library, but there may be some potential change in the composition of the library during amplification due to the different growth rates of the clones. This change in the library clone composition ratio can be minimized by pre-adsorbing the library clones onto bacteria and using a method of high-density plate laying and short-term incubation.

Operation method

basic program

Materials and Instruments

Plasmids
LB Antibiotics
Coated sticks Nitric acid fiber membrane

Move

1. Detection of photocopied colonies on nitrocellulose filtration membranes by in situ hybridization.

2. Positive clones were picked out with a sterile toothpick and inoculated into 1 ml of cold LB medium containing appropriate antibiotics and stored at 4°C to inhibit bacterial growth.

3. Take 1~25 μl of the bacterial suspension and spread it on plates containing the same antibiotic, incubate at 37℃ overnight, and 25~250 colonies should be grown on plates of 100 diameters.

4. Photocopy the colonies on nitrocellulose filter membrane, denature, revert, bake the membrane and hybridize.5. Against the results of the radioautography of the secondary plates, independent positive clones were selected, cultured for amplification and isolated for plasmid or mucoid DNA.

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