Genomic DNA Prep

목차

1. Introduction
2. Materials
3. Method
Reference

본문내용

Introduction
It is the method of choice when large amounts of mammalian DNA are required, for example, for southern blotting or for construction of genomic libraries in bacteriophage λ vectors. Approximately 200 ㎕ of mammalian DNA, 100-150 kb in length, is obtained from 5×107 cultured cells.

Materials
Ammonium acetate (10 M)
Ethanol
Dialysis buffer
50 mM Tris-HCl (pH8.0)
10 mM EDTA (pH8.0)
Lysis buffer
10 mM Tris-HCl (pH8.0)
0.1 M EDTA (pH8.0) -> DNase의 Mg2+ ion을 잡아서 DNase를 inhibit 시킴
0.5% SDS

<중 략>

2. Transfer the lysate to centrifuge tubes.

3. Add proteinase K (20 mg/㎖) to a final concentration of 100 ㎍/㎖. Use a glass rod to mix the enzyme solution gently into the viscous lysate of cells.

4. Incubate the lysate in a water bath for 3 hours at 50℃. Swirl the viscous solution from time to time.

5. Cool the solution to room temperature and add an equal volume of phenol equilibrated with 0.1 M Tris-HCl (pH8.0). Gently mix the two phases by slowly turning the tube end-over-end for 10 minutes.

참고 자료

Sambrook and Russell. 2001. Molecular cloning third edition. vol. 1 : 6.4 - 6.11