Application of bacterial artificial chromosomes

Summary

The bacterial artificial chromosome (RAC ) is a synthetic vector based on the E. coli fertility factor (F). This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Application of bacterial artificial chromosomes

Principle

The bacterial artificial chromosome (RAC ) is a synthetic vector based on the E. coli fertility factor (F).

Materials and Instruments

Restriction endonuclease Escherichia coli culture Electrotransformed receptorized E. coli cells
LB cryobuffer
Pulsed-field gel electrophoresis apparatus LB agar plates LB medium Electroporation apparatus

Move

I. Materials

1. Buffers and solutions

LB cryobuffer

2. Enzymes and buffers

Restriction endonucleases

3. gels

Pulsed-field gel electrophoresis apparatus

4. Culture medium

LB agar plates containing 12.5 μg/ml chloramphenicol

LB medium with 12.5 μg/ml chloramphenicol

5. Specialized equipment

Electroporator

6. Vectors and strains

BAC DNA or BAC isolate transformed E. coli cultures

Frozen electro-transformed susceptible E. coli cells

II. Methods

1. Prepare fresh BAC transformants.

(1) If the BAC is supplied as DNA

(1) Introduce BAC DNA into E. coli (DH10B strain) by electroporation.

② Take 2.5, 25 and 250 μl of the bacterial solution from each batch of electroporation and spread it on LB agar plates containing 12.5 μg/ml chloramphenicol. incubate the plates at 37℃ for 16~24 h.

(2) If BAC is provided in the form of transformed bacterial liquid

① Immediately streak on LB agar plate containing 12.5 μg/ml chloramphenicol.

① Immediately streak on LB agar plates containing 12.5 μg/ml of chloramphenicol.

2. 12 transformed colonies were selected and added into 5 ml of LB medium containing 12.5 μg/ml chloramphenicol respectively, and shaken vigorously at 37℃ overnight.

3. Take 1 bacterial ring from each 12 tubes of 5 ml culture solution and inoculate them into LB freezing medium. After bacterial growth, transfer to -20℃ for freezing and storage.

4. Take 4.5 ml of each 5 ml of culture solution from step 2 and purify BAC DNA.

5. Analyze the BAC DNA.

(1) Identify the target region of BAC containing the chromosome by PCR or by hybridization with SouAem.

(2) Detect the size of the insert fragment by restriction enzyme digestion and PFGE.

6. Based on the results, select one or more BACs for further analysis.

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