The method of DNA extraction of animal tissue cell genome.

, the experimental principle
that all nuclear cells in the ceuteral organism, including
cultured
cells, can be used to prepare genomic DNA. The DNA of the eroteucleotic organism is present in the nuclei of the cell in the form of chromosomes, so the principle of preparing DNA is to separate DNA from
proteins
, lipids and sugars, and to keep the DNA molecules intact. The general process of extracting DNA is to digest and break down proteins in solutions containing SDS (sodium tetranyl sulfate) and protease K, and then extract and separate proteins with phenols and chloroform/isotinol, and the resulting DNA solution is precipitated by ethanol to allow DNA to be extracted from the solution.
properties of protease K are the ability to maintain high activity in the presence of SDS and EDTA (sodium tetamine tetacetic acid). In a reaction system that extracts DNA after homogeneity, SDS destroys cell membranes, nucleic membranes, and isolates tissue proteins from DNA, while EDTA inhibits the activity of Dnase in cells, while protease K degrades proteins into small peptides or
amino acids
, allowing DNA molecules to be completely isolated.
II, Instruments and
Reagents
1. Instruments: ringing temperature
water bath pot, bench-top centrifuge, UV hydrometer (GeneQuant),
piper
, glass homogenizer, centrifuge tube (sterilization), suction head (sterilization) three, operating steps
1. Take fresh or frozen animal tissue blocks 0.1g (0.5cm3), as far as possible. Place in a glass homogenizer, add 1 ml of cell lysate buffer to the missing tissue block, transfer to a 1.5 ml centrifuge tube, add protease K (500ug/ml) 20 l, mix well. Water bath 30min in a heated water bath pot at 65C can also be transferred to a 37C water bath from 12 to 24h, intermittent oscillation centrifuge tube several times. Centrifuge at 12000 rpm centrifuge 5min, take the liquid into another centrifuge tube.
2. plus 2 times the volume of isopropyl alcohol, after reverse mixing, you can see the silk, with 100ul suction head pick out, cool dry, with 200ul TE re-dissolved. (
PCR
reaction, etc., further purification is required in the following steps)
3. Add the same amount of phenol: chloroform: isosterol oscillating mix, centrifugation 12000 rpm, 5min.
4. Take the upper solution to another tube and add chloroform of equal volume? Isosterol, oscillating mix, centrifugal 12000 rpm, 5min.
5. Take the upper solution to another tube, add 7.5mol/L acetaminophen of 1/2 volume to add 2 times the volume of aqueous ethanol, mix and mix the room temperature precipitation 2min, centrifuge 12000 rpm, 10min.
. Carefully remove the liquid, pour the centrifugal tube on the absorbent paper, and remove the residual droplets attached to the pipe wall.
7. Wash the sediment 1 time with 1 ml 70% ethanol, centrifuge 12000 rpm, 5min.
8. Carefully pour out the liquid, pour the centrifugal tube on the absorbent paper, remove the residual droplets attached to the pipe wall, and dry the
room
..9. Add 200ul TE to re-dissolve the sediment and place it at 4 degrees C or? Save the standby at C20 degrees C.
10. Take the appropriate amount of samples to detect concentration and purity on GeneQuant.
, FAQs,
.1. The selected experimental materials should be fresh and the processing time is not easy to be too long.
2. Before adding a cell lysate buffer, the cells must be evenly dispersed to reduce DNA lump formation.
3. The extracted DNA is not easily dissolved: it is imperculsible and contains more impurities; The sediment is too dry and will make it difficult to dissolve.
. DNA is coated during electrophoresis testing: careless operation; contamination of nucleases, etc.
5. A280/A260 of the hydrolight analysis DNA is less than 1.8; In this case, SDS should be added to the final concentration of 0.5%, and repeat steps 2 to 8.
6. After phenol/chloroform/isosterol is extracted, the liquid is too mucusy to absorb: high concentrations of DNA can increase the amount of pre-pumped buffer or reduce the amount of tissue taken.
.