DNA preparation of salmon sperm stretcher

Summary

Using this scheme, well-sheared, high-quality salmon sperm stretcher DNA can be obtained, which can be used in transformation experiments and other experiments that require high-quality stretcher DNA.

Operation method

basic program

Materials and Instruments

Salmon Sperm DNA
TE Phenol Chloroform Sodium acetate
Centrifuge Shaker Ultrasonic

Move

1. Add TE buffer to a beaker containing dried salmon DNA until the concentration of salmon DNA is 5-10 mg/ml. blow repeatedly with a 10 ml pipette and stir with a stirring bar at 4°C overnight to obtain a homogeneous viscous liquid.
2. A larger ultrasonic probe is inserted into a beaker of liquid and the DNA is sheared by ultrasound to DNA fragments of 2-15 kb (average 7 kb) in size.

3. Extract with buffer-equilibrated phenol. Transfer the sheared salmon sperm DNA solution to a 50 ml conical tube, add an equal volume of buffer-equilibrated phenol, mix well, and centrifuge at 3,000 g for 5 to 10 min (or until clearly phased). The upper phase containing the DNA is transferred to a clean test tube.

4. Equilibrate phenol/chloroform and chloroform with 1:1 (v/v) buffer and extract separately, then transfer the upper phase containing DNA to a centrifuge tube suitable for high speed centrifugation.5. Add 1/10 volume of 3 mol/l sodium acetate and 2.5 volumes of ice-cold anhydrous ethanol, mix thoroughly and centrifuge at approximately 12,000 g for 15 min.6. The supernatant is discarded, the precipitate is washed with 70% ethanol and dried, and the DNA is reconstituted in sterile TE buffer to a concentration of 5~10 mg/ml.7. Denature the DNA at 100°C for 20 min, then quickly place in an ice bath. Dispense DNA in microcentrifuge tubes and store at -20°C, remove and thaw when needed, and reboil before using for transformation experiments.

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