真核基因组DNA的提取原理、提取操作、鉴定在线视频

DNA extraction is one of the most fundamental and essential echniques

in the study of DNA.

In this video,

we will show you how to extract DNA from mouse liver cells.

DNA extraction is the isolation and purification of DNA from cells.

First, cells are lysed to release the DNA into solution. 

The detergent SDS (Sodium Dodecyl Sulfate)

is often added to dissolve the cell membrane and disrupt proteins,

helping release the DNA from the nucleus and cell.

Next the DNA must be separated from other cellular molecules,

such as proteins and RNA.

Proteinase K and RNase A are added

during lysis to degrade proteins and RNA respectively,

leaving the DNA intact.

DNA can be purified through Column binding,

which will remove a bulk of the soluble impurities.

Salts are added during DNA extraction to stabilize the DNA

and assist in precipitating it out of solution.

Washing steps are performed with Wash Buffer

(add ethanol before using)

to remove residual impurities from the bound DNA.

Finally, Genomic DNA is eluted with Elution Buffer low in salt. 

Step 1: Sample preparation

A mouse is sacrificed.

The liver is removed immediately,

cut into small pieces with a pair of scissors,

grinded with a mortar and pestle.

Transfer 20-30mg homogenate to a 1.5ml microtube.

Step 2: Cell lysis

Add 600μl Lysis Buffer A,

mix well by inversion of tube.

Add 10μl Proteinase K,

incubate for 1h at 60 ℃,

mix well by inversion of tube 4-6 times during incubation.

Add 10μl RNase A,

incubate for 10 min at room temperature.

Add 400μl Lysis Buffer B,

mix thoroughly by vortex for 30 sec,

centrifuge at 12,000 rpm for 5 min.

Step 3: DNA binding

Carefully transfer 750μl supernatant to a Spin Column,

place for 2 min,

centrifuge at 12,000 rpm for 1min,

discard the flow-through.

Step 4: DNA wash

Add 700μl Wash Buffer A,

centrifuge at 12,000 rpm for 1 min,

discard the flow-through.

Add 700μl Wash Buffer B,

centrifuge at 12,000 rpm for 1 min,

discard the flow-through.

Add 500μl Wash Buffer B,

centrifuge at 12,000 rpm for 1 min,

discard the flow-through.

Centrifuge at 12,000 rpm for 2 min,

Place the Spin Column on a clean 1.5ml tube

air dry DNA pellet to remove ethanol.

Step 5: DNA elution

Add 30μl Elution Buffer

(preheat to 55～65 ℃ before using),

Incubate at room temperature for 2 min,

centrifuge at 12,000 rpm for 2 min.

Genomic DNA is eluted from the Spin Column into Elution Buffer.

Integrity of DNA can be checked by 1% agarose gel electrophoresis.

Concentration and purity of DNA can be checked by spectrophotometer.