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 Glycerol
Nitrocellulose membrane Incubator

Move

1. Add a layer of nitrocellulose membrane to the antibiotic-containing plate, spread the resistant bacteria on the nitrocellulose membrane, and incubate the bacteria until the edges of the colonies just meet.

2. Add LB culture solution to the plate with full colonies, about 2 ml for a 10 cm plate and 4 ml for a 15 cm plate, and use a sterilized cell scraper to scrape the colonies from the nitrocellulose membrane to form a bacterial suspension.

3. Collect the bacterial suspension from the plate into a 50 ml plastic tube, add sterile glycerol to a final concentration of 15%, mix thoroughly, and then dispense it into 1 ml tubes, 500 μl per tube, and freeze it at -70℃ (small portions thus dispensed can be stored for more than one year and can still be viable).4. To screen the library, a frozen tube is removed, thawed and titrated for bacterial concentration, and then the bacteria are spread on the screened filter membrane.

Caveat

The most important concern with any amplification step is that each initial recombinant should have an equal chance of being amplified, and if the recombinants have different rates of proliferation, this will result in over- or under-representation of the amplified library.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/