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

Phage
MgSO4 Maltose Agarose Chloroform DMSO SM
Centrifuge Spectrophotometer Water Bath

Move

1. Preparation of plate spreading bacteria

(1) 2.5 ml of fresh overnight culture of host bacteria was inoculated in 250 ml of LB culture medium containing 0.2% maltose and 10 mmol/l _MgSO4.

(2) Shake vigorously in a constant temperature shaker at 37℃ for 2~4 h. The concentration of bacterial suspension in the culture medium reached about 0.5 _OD600.

2. slightly centrifuge the microcentrifuge tube containing the library on a centrifuge in order to separate it from chloroform.

3. Add the packed and titrated phage aqueous phase (without chloroform) to the plate-laying bacteria and incubate at 37℃ for 15 min.4. Add 0.5 ml of phage/host bacterial mixture to 8 ml of 47°C warmed top agarose, mix well, and pour quickly onto a fresh 37°C warmed 150 mm H flat pole.

5. Gently rotate the plate and let the top layer of agarose spread well, then leave it for 5 min to solidify, then invert the plate and incubate it at 37℃ for 6-7 h.6. Remove all transfected plates from the thermostatic incubator, add 10 ml of SM suspension medium to each plate separately, and leave at 4℃ for 2~16 h.

7. Elute the phage, then collect SM from all plates and combine into a glass vial or polypropylene plastic tube.

8. Centrifuge at 2,800 g for 5 min, transfer the supernatant to a new glass tube with a Teflon cap, add 0.5 ml of chloroform, mix well, and titrate the amplified library.

Common Problems

Corresponding E. coli host strains used to amplify λ vector libraries

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/