Until the mid-1980s, there were no techniques for isolating, amplifying, and analyzing large sticky fragments of genomic DNA. Prior to that time, if a DNA fragment was to be studied in detail for physical and functional purposes, the length of the DNA should be such that it could be loaded into λ phage particles or sticky vectors. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Application of yeast artificial chromosomes

Principle

Until the mid-1980s, there were no techniques for isolating, amplifying, and analyzing large sticky fragments of genomic DNA. Prior to that time, if a DNA fragment was to be studied in detail for physical and functional purposes, the length of the DNA should be such that it could fit into a λ-phage particle or a sticky vector.

Materials and Instruments

Brewer's yeast
Glycerol
Pulsed Field Gel Electrophoresis Instrument Selective Medium YPD Agar Plate YPD Medium Test Tube

Move

I. Materials

1. Buffers and solutions

30% (V/V) glycerol prepared in YPD medium

2. gels

Pulsed-field gel electrophoresis apparatus

3. Culture medium

Selective medium

YPD agar plates

YPD Medium

4. Specialized equipment

Test tube (2 ml )

5. Carriers and yeast strains

Saccharomyces cerevisiae carrying a recombinant YAC clone

II. METHODS

1. Immediately after obtaining the clone in the laboratory, inoculate the clone by streaking in a selection medium and incubate at 30°C for 48 h to obtain a single colony.

2. 6~12 single colonies were picked and added into 10 ml of YPD medium. Incubate the cultures at 30℃ with vigorous shaking (300 r/min) overnight. During this period, the bacterial count should reach saturation ( _OD600=2.0~3.0, about 3 X ¹⁰⁷ cells/ml).

3. 9 ml of each culture was taken separately and yeast DNA was extracted.

4. Analyze the size of the YAC in each DNA product by PFGE.

5. The presence of target sequences in the YAC DNA is determined by Southern hybridization or PCR.

6. If the results are satisfactory, i.e., the cultures contain YACs of the same size, with no evidence of instability or rearrangement, and the target sequence is present, one or two cultures can be selected for long-term storage.

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