DNA extraction 이론 및 방법

소개

다양한 실험 샘플에 따라 DNA를 추출하는 방법에 대한 내용과 실험 tip 뿐 아니라
troubleshoting 을 정리해 놓은 ppt

목차

1. DNA isolation
Outline of a basic DNA Extraction
Alkali lysis method
Eukaryotic genomic DNA extraction from cell line
Eukaryotic genomic DNA extraction from tissue
Eukaryotic genomic DNA extraction from paraffin block
DNA Quantitation & Quality 측정
TROUBLESHOOTING

본문내용

Outline of a basic DNA Extraction

>>DNA extraction시 RNA와 protein을 제거하지 않으면 나중에 restriction enzyme treatment나 sequencing 반응에 영향을 미칠 수 있기 때문에 intact 한 DNA extraction 이 중요함

1) Break open (lyse) the cells containing the DNA
Physical method-by sonicating, bead beating or vortexing
Chemical method-phenol is often effective for breaking down protienacious cellular walls or viral capsids. Detergent such as SDS is often necessary to remove lipid membranes

2) Protein 제거
Cellular proteins may be degraded with the addition of a protease. When the sample is vortexed with phenol-chloroform and centrifuged the proteins will be seperated.

3)DNA precipitation
Soluble DNA is the precipitated by mixing with cold ethanol or isopropanol and then centrifuging.

4)Washing & Elution
The 70% alcohol serves as a wash to remove the salt previously added. the DNA can be re-suspended in a buffer such as TE or D.W.

5)DNA confirm
Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide and checking under UV light

참고자료

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