Knowledge Sharing: Animal Cell Genomic DNA Extraction

Experimental principle

Eukaryotic DNA is present in the nucleus in the form of chromosomes. DNA is prepared to separate DNA from proteins, lipids, and sugars while maintaining the integrity of the DNA molecule. The process of extracting DNA refers to digesting and decomposing protein in a solution containing dispersed SDS (sodium dodecyl sulfate) and proteinase K, and then separating the protein by extraction with phenol and chloroform/isoamyl alcohol, and obtaining the DNA. The solution was precipitated by ethanol to precipitate DNA from the solution. An important property of proteinase K is its ability to maintain high activity in the presence of SDS and EDTA (disodium edetate).

In the reaction system for extracting DNA, SDS is used for cell membrane, nuclear membrane, and tissue protein and DNA are separated, EDTA inhibits Dnase activity in cells; and proteinase K can degrade protein into small peptide or amino acid to make DNA molecule Separate.

Experimental Materials

(1) Cell lysis buffer:

Tris (pH 8.0) 100 mmol/L; EDTA (pH 8.0) 500 mmol/L; NaCL 20 mmol/L; SDS 10%; pancreatic RNase 20 ug/ml.

(2) Protease K: 20 mg of proteinase k was weighed and dissolved in 1 ml of sterilized double distilled water, and sterilized by filtration through a disposable filter, and stored at -20 ° C for use.

(3) TE buffer (pH 8.0), stored at room temperature after autoclaving.

(4) Phenol: chloroform: isoamyl alcohol = 25:24:1 (volume ratio).

(5) NaAc 3 mol/L.

(6) Isopropyl alcohol, cold anhydrous ethanol, 70% ethanol, and sterilized water.

experiment procedure

1. Take fresh or frozen animal tissue block 0.1g (0.5cm3) and cut it as much as possible. Place in a homogenizer, add 2 ml of cell lysis buffer, and then grind thoroughly until no obvious tissue blocks are visible, then transfer to a 1.5 ml centrifuge tube and centrifuge at 12000 rpm for 5 min.

2. Discard the supernatant, add 0.4 ml of cell lysis buffer, and quickly disperse the pellet. 20 ul of proteinase K (0.2 mg/ml) was added and mixed. The water bath was placed in a 65 ° C constant temperature water bath for 30 min, or transferred to a 37 ° C water bath for 12-24 h, and the tube was shaken intermittently several times. Centrifuge at 12000 rpm for 5 min in a bench top centrifuge and take the supernatant into another centrifuge tube.

3, add an equal volume of phenol: chloroform: isoamyl alcohol extraction once, gently mix. Centrifuge at 10,000 rpm for 10 min at 4 °C.

4. Gently draw the upper liquid level, taking care not to absorb the white precipitate between the two layers. The white precipitate in the liquid level of the above layer may be more or more turbid, and an equal volume of phenol may be added again: chloroform: isoamyl alcohol is extracted once.

5. Add 1/10 volume of NaAc to the aspirated supernatant, mix gently, then add 2.5 volumes of pre-cooled absolute ethanol at -20 ° C, gently mix, and precipitate in a refrigerator at -20 ° C overnight.

6. Centrifuge at 12 000 rpm for 20 min, discard the supernatant; add 75% ethanol pre-cooled at -20 °C, centrifuge at 12000 rpm for 15 min. Warm the chamber, add appropriate amount of sterile water to dissolve the genomic DNA, store at 4 ° C, if not dissolved enough Shake and dissolve overnight.

Precautions

1. The selected organization must be fresh material and the processing time should not be too long.

2. The operation process should be carried out under low temperature conditions as much as possible to avoid degradation or fragmentation of genomic DNA.

3, the use of phenol: chloroform: isoamyl alcohol, ethanol extraction or precipitation of DNA must be gently operated to avoid DNA degradation or breakage.

4. Organic reagents will be used in the experiment, which must be protected and operated under ventilated conditions.

Common problems and analysis

Remarks: Weizheng Biotechnology Co., Ltd. is committed to providing high quality virus packaging services for researchers. Services include: scientific and clinical grade adenovirus, lentivirus, adeno-associated virus (AAV) packaging, plasmid vector construction, TALEN gene Knockout, gene mutation, etc. So far, the company has a human-source stock database library (18,000), an adenovirus stock library (12,000), and an adeno-associated virus (AAV) stock library. The company also has a wealth of customized services. Sincerely welcome you to consult and purchase!