The application of a modified version of the salt precipitation method of Miller et al. (1988) for DNA purification, which does not use phenol or chloroform, is milder and effective in preventing breakage of long chromosomal DNA. Another method of DNA purification is described in Chapter 9. This method precipitates nuclei and has been used successfully to purify DNA from blood samples, cultured cells, and breast tumor tissue.
DNA purification. This experiment was derived from PCR Laboratory Guide (Second Edition) by Kang Seed and Lijia Qu.
Operation method
High-quality DNA purification assay
Materials and Instruments
Breast Tumor Tissue Move I. Materials For more product details, please visit Aladdin Scientific website.
EDTA Isopropyl Alcohol Lysis Buffer Nucleus Lysis Buffer Triton Lysis Buffer
Water bath Glass rod
1. Buffers, solutions and reagents
EDTA, 100 mmol/L
Isopropyl alcohol
Lysis buffer
10 mmol/LTris-HCl
1 mmol/LEDTA
150 mmol/LNaCl
0.5% SDS, pH 10.5
NaCl, 0.15 mol/L and 6 mol/L
Nucleus lysis buffer
75 mmol/L NaCl
24 mmol/LEDTA,pH8.0
Proteinase K or streptavidin, 20 mg/ml.
SDS,200 g/L
Sucrose, lmol/L
Tris-HCl,2mol/L,pH9.2
Triton lysis buffer
10mmol/L Tris-HCl
5 mmol/L,MgCl2
1%TritonX-100
TE buffer (lOmmol/L Tris-HCl, lmmol/LDTA, pH 7.5)
Sucrose, pH 9.2
TritonX-100
2. Specialized equipment
Water bath, preheated to 55°C
Glass rod
3. Cells and tissues
Treated breast tumor tissue (100 mg tissue for hormone receptor analysis) or 5X105 cultured cells washed twice with PBS.
Methods
1. Add 750ul of Lysis Buffer and 200ug of Proteinase K (or Streptavidin) to a l0 ml tube containing cells; hold at 55°C for at least 90 min until the precipitate is dissolved.
2. If the precipitate does not dissolve, repeat step 1.
3. Add 250ul of 6mol/L NaCl and shake carefully.
4. centrifuge (3000r/min) at 4°C for 15 min. carefully transfer the supernatant to another new tube.
5. Repeat steps 3 and 4.
6. Add an equal volume of isopropanol and invert the tube until the DNA is visible. wrap the DNA around a glass rod, gently pull the DNA out along the wall of the tube, and transfer it to 300ul of TE or sterilized double-distilled water.
7. Place at 4°C and allow to dissolve.
Extract DNA from 10 ml of blood
8. Add 40 ml of pre-cooled (4°C) Triton Lysis Buffer.
9. Place at 4°C for 30 min; turn tubes over every few minutes.
10. Centrifuge (3000 r/min) for 30 min at 4°C and discard supernatant.
11. Wash the precipitate (nuclei) with 5-15 ml of 0.9% (0.15mol/L) NaCl.
12. Centrifuge at 2300r/min for 10min.
13. Discard the supernatant. Cell nuclei can be stored at -20°C.
14. Add 3 ml of Nucleus Lysis Buffer and shake well.
15. Add 25ul Streptavidin (20 mg/ml) and 230ul 10% SDS and shake at room temperature overnight.
16. Add 1 ml of 6mol/L NaCl and shake well.
17. Centrifuge (3000r/min) at 4°C for 15 min and carefully transfer the supernatant to another new tube.
18. Repeat step 10.
19. Add an equal volume of isopropanol and turn the tube upside down until the DNA is visible. wrap the DNA around a glass rod, gently pull the DNA out along the wall of the tube, and transfer it to 400ul of TE or sterilized double-distilled water.
20. Place at 4°C and allow to dissolve.