Preparation of cloning experiments

Summary

This experiment describes the method of PCR product preparation for cloning. This experiment is derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

Preparation of cloning experiments

Materials and Instruments

ATP - mercaptoethanol IPTG X-gal flat-end restriction enzyme ligation buffer T4DNA ligase flat-end insert Cloning vector Culture medium
FALCON 2059 Multi-polypropylene tubes 37°C Thermostat Water baths

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I. Materials

1. Buffers, solutions and reagents

ATP (10 mmol/L)

β-mercaptoethanol (optional)

IPTG (100 mmol/L, dissolved in water)

X-gal (100 mg/ml, dissolved in dimethylformamide)

2. Enzyme and enzyme buffer

Flat-end restriction enzymes (5U), e.g., Srf I restriction endonuclease (Stratagene)

Ligation buffer, l0X (250 mmol/L Tris-HCl, pH 7.5, 100 mmol/L MgCl2, 100 mmol/LDTT, 200ug/ml BSA)

T4DNA Ligase (4U)

3. Nucleic acids and oligonucleotides

Flat End Insert (100ng/ul)

Cloning vector (10ng/ul)

4. Culture medium

(1) SOC medium (per liter: 20 g tryptone, 5 g yeast extract, 0.5 g NaCl, add water to 900 ml. Autoclave. In addition, the following were mixed separately: 2.03 g magnesium chloride, 1.2 g magnesium sulphate, 3.6 g glucose, added to water to a final volume of 100 ml, filter sterilized. (Then add the cooled autoclaved medium).

(2) LB (Luria Broth) Spheroplate [Amount per liter: lOg NaCl, lOg Bacto-tryptone, 5 g Bacto-yeast extract, 20 g Bacto agar. Adjust pH to 7.0 with 5 mol/L NaOH. add deionized water to a final volume of 1 L. Autoclave and pour into petri dishes (approx. 25 ml/100 mm plate)].

(3) Penicillin-neo-penicillin LB plates (20ug/ml penicillin, 80ug/ml neo-penicillin). Used to reduce the formation of satellite colonies. Autoclaved medium was cooled to 55°C, and filter-sterilized antibiotics were added.

(4) Chloramphenicol LB plate (30ug/ml).
Autoclaved medium was cooled to 55°C and filter-sterilized antibiotic was added.

(5) X-gal/IPTG plates are optional: to phenotypically select for transformants, add 20ul of X-gal and 20ul of IPTG solution to an agar plate containing the appropriate antibiotics and shake immediately to distribute them evenly (slight precipitation may occur).
Do not mix X-gal and IPTG, as these compounds will precipitate.
Important: Allow the plate to dry for 15-30 min before coating.

5. Specialized equipment

FALCON 2059 Multi-polypropylene Tubes

37°C thermostat

Water Bath, Preset to 37°C, 42°C, 65°C

6. Cells and tissues

Appropriate strain of Escherichia coli, e.g., XLl-Blue (Stratagene), made into a sensory state according to the method described by Sambrook and Russe II (2001), or go for well made ones.

II. Methods

1. Connection

(1) Set up the PCR-Script reaction system in an autoclaved 1.5 ml test tube and add the following reagents in order.

Cloning vector (10ng/ul) 1ul

Ligation buffer, 10X 1ul

ATP (10 mmol/L) 0.5ul

Pfu light-polished PCR product insert 1 to 4ul

Srf I restriction endonuclease (5U) 1ul

T4DNA ligase (4U) 1ul

_H2O added to 10ul

~undefinedFor ligation reactions, the ideal insert:vector DNA value is variable. For sample DNA, from 5:1 (when using a polished insert) to 100:1 (when using a non-polished insert) may be necessary. Larger insert:vector values are required when using non-polished inserts, due to the low chance that both ends of a PCR fragment will be flat-terminated. For a particular insert fragment, it is best to optimize the conditions using the following equation:

pmol ends/ugDNA = ^2X106/number of base pairs

(2) Mix gently and warm bath at room temperature for 1~2 h.

(3) Heat treat the sample at 65°C for 10 min.

(4) Store the samples on ice and prepare for transformation of receptive E. coli.

2. Transformation

(1) Melt the receptor cells on ice.

(2) Mix the cells with gentle stirring and remove 40ul of cells into a 15 ml pre-cooled FALCON 2059 tube.

(3) Optionally, add ethanol to 40ul of bacteria (to a final concentration of 25 mmol/L).

(4) Stir gently and place on ice for 10 min, stirring gently every 2 min.

(5) Add 2ul of heat-treated ligation product DNA (step 4, cloning procedure).

(6) Place on ice for 30 min.

(7) Heat shock in a water bath at 42 C for 30 s. The length of the heat shock is critical for maximum efficiency.

(8) Place the conversion mixture on ice for 2 min.

(9) Add 450ul of preheated (42°C) SOC medium and incubate for 1h at 37°C with 225~250r/min oscillation.

(10) Remove 50~200ul (100ul is the standard amount) of the transformed mixture and spread it evenly on the appropriate antibiotic plate with a sterile fork.

Optionally: add a substrate to the LB plate that generates a pigment to detect recombinants; also see β-galactosidase color selection, below.
If applying 100ul of shaken culture, the cells can be spread directly onto the plate; if applying less than 100ul of transformation mixture, increase the final volume of transformation mixture to 200ul with SOC medium, and then spread the plate.

(11) Incubate the plate at 37°C overnight (approximately 16 h).

(12) Select the white spots for detection, do not select clones that have a slight blue color or are blue in the center.
Colonies containing inserts are initially white and may develop a slight blue color after 2 to 5 days in the petri dish.

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