Recombinant phage libraries can be amplified by plate culture, and the original seed for plate culture can be derived directly from the packaging mixture described in this protocol. Wherever possible, however, this amplification process can be omitted and the DNA sequences of interest can be screened directly from the original library. Amplification will inevitably reduce the complexity of the library, in part because it is highly detrimental to slow-growing recombinant phages in successive rounds of phage growth. The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" Translated by Huang Peitang et al.

Operation method

Amplification experiments of genomic libraries

Principle

Recombinant phage libraries can be amplified by plate culture, and the original seed for plate culture can be derived directly from the packaging mixture described in this protocol. Wherever possible, however, this amplification process can be omitted and the DNA sequences of interest can be screened directly from the original library. Amplification will inevitably reduce the complexity of the library, in part because it is highly detrimental to slow-growing recombinant phages in successive rounds of phage growth.

Materials and Instruments

λ phage library Escherichia coli spread trigger bacteria
Chloroform SM
LB or NZCYM Agar plates Sorvall SS-34 or equivalent Heating device or water bath

Move

I. Materials

1. Buffers and solutions

Chloroform

SM

2. Culture medium

LB or NZCYM agar plates (150 mm)

LB or NZCYM top agarose

3. Centrifuges and rotors

Sorvall SS-34 or equivalent

4. Specialized equipment

Heating device or water bath pre-set at 47°C (for top agarose layer)

5. Carriers and strains

λ Phage libraries

Escherichia coli spreading trigger bacteria

II. METHODS

1. In order to amplify the λ phage library, mix 50 μl or less volume of the multiple packaging mixture component containing 10,000~20,000 recombinant phages with 0.2 ml of flat-breaded bacteria in a 13X100 mm test tube, and incubate at 37℃ for 20 min.

2. Add 6.5 ml of melted 47°C agar or agarose to the first tube of infected cells, mix by tapping or gentle vortexing, and immediately pour onto the bottom layer of agar in a freshly filled 150 mm plate. The procedure was repeated for the remaining infected cells.

3. Allow the plate to stand at 37°C for a maximum of 8-10 hours.

4. Add 12 ml of SM to the plate (150 ml if using a baking tray) and place horizontally at 4°C overnight.

5. Collect the SM from all plates into a separate sterile polypropylene centrifuge tube or bottle. Rinse each plate with an additional 4 ml of SM, combining it with the initially recovered SM solution. Add 0.2 ml of chloroform to the recovered amplified phage culture and allow to stand at room temperature for 15 min with occasional gentle shaking to allow the chloroform to lyse all infected cells.

6. Centrifuge at 4000 g (5800 r/min in Sorvall SS-34) at 4°C for 5 min to remove cell and agarose debris.

7. Transfer the supernatant to a new sterile tube or bottle. Separate the amplified phage library into several fractions and store at 4℃. Determine the titer of the library by phage spot analysis.

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