[실험노트] 분자생물학 실험 2. DNA 추출 Genomic DNA extraction 실험보고서

Title : Genomic DNA extraction

 

Instruction :

1. Purpose

- To understand procedures to extract bacterial genomic DNA with physical lysis method

- To understand the background of methods to quantify DNA

 

2. Principle :

1) In this experiment, we would like to use a physical lysis method (with glass beads) for bacterial DNA extraction from unknown bacterial strains that are not well extracted by chemincal lysis method.

Suggest reasons why chemical lysis is not well worked on this bacterial strain

이 실험에서 우리는 화학적 방법으로의 용해가 어려운 미지의 bacterial strains으로부터 DNA추출을 위해 glass beads를 이용한 물리적 세표용해 방법을 사용한다. bacterial strain의 화학적 용해가 어려운 이유가 무엇일까

=> peptidoglycan 층이 두껍기 때문

 

2)Suggest the proper size of beads for this experiment

이 실험에서 적절한 glass beads의 크키는?

=> 40-400um

 

3) DNA 측정 방법

DNA quantitation 의 두 방법으로는 fluorescence(형광발광체)를 이용한 Qubit 과 UV 가시광선 흡수를 이용한 Nanodrop 나노드롭 방법이 있다. 두 방법 매우 적은 시료를 필요로 하며 모두 정확하고 쉽고 빠른 biomolecules의 quantitation이 가능하다.

먼저 Qubit은 특이적인 biomolecules에 부착하는 dye를 사용한다. DNA, RNA, Oligos 등을 포함한 mucleic acid 의 총량을 측정한다.

Nanodrop은 DNA, RNA, protein 의 순도 측정에 사용되는데, 260nm에서의 흡수율 측정으로 농도를 알아낼 수 있다. 기기에서 흡수 스펙트럼으로 나타내어 순도 혹은 불순물 존재에 대한 정보를 제공한다.

 

4) Assuming that the DNA obtained in this experiment shows a high A260/A280 ratio, then suggest how you can improve this

=> 이번 실험에서 얻은 DNA가 높은 A260/A280 값을 나타낸다고 가정할 때, 어떻게 이것을 개선할 것인지

 

 

Abnormal 260/280 ratios usually indicate that the sample is either contaminated by protein or a reagent such as phenol or that there was an issue with the measurement.

A low A260/A280 ratio may be caused by:

• Residual phenol or other reagent associated with the extraction protocol

• A very low concentration( > 10 ng/ul).of nucleic acid

High 260/280 purity ratios are not indicative of an issue.

Although purity ratios and spectral profiles are important indicators of sample quality, the best indicator of DNA or RNA quality is functionality in the downstream application of interest. If the purity ratio is significantly higher than expected, it is best to review the spectral profile as a primary means of troubleshooting. It is important to note that there are occasions when the purity ratios are within expected limits, yet there is a problem with the sample. 

 

Ration > 1.9 can be caused by RNA contamination. Usually, RNA contaminatin do not interfere with downstream application. Depending on sample type, amount, and disruption procedure, prepatations might contain small amounts of RNA. If it is necessary to reduce RNA contamination to the lowest possible level, incubate the lysate after the distruption step for 5min at 70'c in order to incubate the Proteinase K. After cooling to romm temperature, add 20ul RNase A (20mg/ml) and incubate 5min. Continue with the application of the lysate onto the column.

 

Materials :

Incubated bacteria (unknowm), latex gloves, markerpen, pipet (1ml, 200ul, 10ul), tip (1ml, 200ul, 10ul), eppen tubes, eppen tube rack, nucleospin, microbial DNA kit, centrifuge machine, swing mill, vortex mixter, nanodrop

 

Method :

1. Prepare sample

(40mg microbial pellet (wet weight) 100ul BE

2. Lyse sample

Transfer sample in Nucleospin Bead. Tube type B

40ul Buffer MG

10ul Liquid Proteinase K

Agitate on a swing mill or similar device, 4-12min

11,000 xg, 30s

3. Adjust binding conditions

600ul Buffer MG

vortex 3s

11,000xg, 30s

4. Bind DNA

Load 500-600ul sample on NucleoSpin microbial DNA column

11,000xg, 30s

5. Wash silica membrane

1st 500ul BW. 11,000xg,30sㅇ

2nd 500ul B5. 11,000xg, 30s

6. Dry silica membrane

11,000xg, 30s

7. Elute DNA

100ul BE

RT, 1min

11,000xg, 30s

 

DNA quantitation method DNA추출 실험 방법 :

1) Nano drop 설정

- (DNA, ds DNA)

2) Blank 설정

- Elution buffer or autoclave 3'dw 사용

- 1.5 -2 ul을 동그란 볼에 기포 없이 놓아둔다.

- 뚜껑을 닫고 blank 설정 버튼을 눌러준다

- Kim tech 을 이용하여 제거

- 한번 더 elution buffer 나 autoclaved 3'dw 1.5ul-2ul을 이용하여 blank 설정함

3) DNA 농도 및 순도 측정

- Kim tech 이용해서 측정하는 곳 닦아준 후 측정할 시료 1.5-2ul을 놓아준다.

- 뚜껑을 닫고 measure 버튼을 눌러준다.

Nano drop을 이용해 확인해야 할 수치

1. A260/A280 ration

2. DNA quantity (ng/ml)

 

 

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