The freeze-thaw method of recovering DNA fragments can be used to (1) obtain purified DNA fragments and (2) recover PCR products.
Operation method
Recovery of DNA fragments by freeze-thawing
Principle
Freezing and thawing method refers to the use of repeated freezing and thawing due to the formation of ice crystals in the cells and the increase in the concentration of salt in the remaining liquid can rupture the cells. This method is simple and convenient, but it should be noted that those proteins that are sensitive to temperature changes should not use this method.
Materials and Instruments
DNA Glue Strips Move 1. carefully cut off the adhesive strip containing the DNA to be recovered under UV light, mash the cut strip (less than 0.6 g) and place it in a 1.5 ml centrifuge tube; 2. Add an equal volume of Tris-HCl saturated phenol (pH 7.6) and mix well with shaking; 3. -20℃ for 5-10min; 4. Centrifuge at 10,000g x 5min at 4°C, and transfer the upper layer into another centrifuge tube; 5. Add 1/4 volume of H2O to the centrifuge tube containing the gel and mix well with shaking; 6. -20℃, leave for 5-10min; 7. Centrifuge at 10,000g x 5min at 4°C and combine the supernatants; 8. Extract the supernatant once with equal volume of phenol/chloroform and chloroform respectively; 9. Add 1/10 volume of 3M NaAc (pH 5.2), 2.5 times volume of pre-cooled anhydrous ethanol, mix well; 10. -20℃, let stand for 30min; 11. Centrifuge at 13000g x 10min at 4°C, discard supernatant, wash the precipitate 1-2 times with 75% ethanol and dry; 12. Add appropriate amount of H2O or TE to dissolve the precipitate. Caveat The adhesive strip of DNA should be melted all the way down before extraction. Common Problems Nowadays, kits are mostly used for DNA recovery. For more product details, please visit Aladdin Scientific website.
Tris-HCl saturated phenol H2O phenol chloroform chloroform anhydrous ethanol NaAc TE
Centrifuge Tubes Centrifuge