Genomic DNA preparation in mammals
Genomic DNA preparation in mammals
Various factors that break and degrade DNA should be avoided as much as possible during DNA extraction to ensure the integrity of DNA and lay the foundation for subsequent experiments. Generally eukaryotic genomic DNA has 107-9 bp and can be extracted from fresh tissues, cultured cells or cryopreserved tissue cells, often achieved by digesting the cells with proteinase K in the presence of reagents such as EDTA as well as SDS, followed by phenol extraction.
Operation method
Preparation of DNA
Materials and Instruments
Animal Tissue Move 1. Cut off the tissue and immediately cut into small pieces and freeze in liquid nitrogen.2. 200 mg to 1 g of tissue is crushed in a pre-cooled mortar and pestle, or pounded to a fine powder with a hammer, and suspended in 1.2 ml of digestive buffer per 100 mg of tissue. Caveat 1. If liver tissue is used, remove the gallbladder as it contains high levels of various digestive enzymes. For more product details, please visit Aladdin Scientific website.
PBS Ammonium acetate Ethanol Phenol Chloroform Isoamyl alcohol TE
Centrifuge Shaker Mortar
3. Centrifuge the cells at 500 g for 5 min and discard the supernatant. The adherent growth cells were first digested with trypsin.
4. Resuspend cells in 1-10 ml of ice-cold PBS, centrifuge at 500 g for 5 min, discard supernatant, and repeat. 5.
5. Resuspend cells with 1 volume of digestion buffer.6. The samples are incubated in tightly capped tubes at 50°C for 12-18 h with shaking.7. Extract the sample with an equal volume of phenol/chloroform/isoamyl alcohol and centrifuge at 1,700 g for 10 min. 8.
8. If the sample does not dissolve well, add 1 more volume of digestion buffer without protein spiking K and repeat centrifugation.
9 If there is a thick layer of white material at the interface, repeat the organic extraction and transfer the upper layer (aqueous solution) to a new tube.10 Add 1/2 volume of 7.5 ml ammonium acetate and 2 volumes of 100% ethyl alcohol and centrifuge at 1,700 g for 2 min.11. Wash with 70% ethanol, air dry and precipitate with TE.
12. Redissolve in buffer to give a final concentration of around 1 mg/ml.