DNA 분리 및 제한 효소 분석
At the end of this lab, students should know...
What are the essential steps of DNA isolation?
The essential steps of DNA isolation are cell harvesting, lysis, protein degradation, and DNA precipitation.
How is the quantity and purity of DNA determined?
Scientists use a spectrophotometer, which measures the intensity of a light beam passed through a sample as a function of its wavelength. By quantifying the output of wavelengths that are specific to nucleic acids or impurities, one can not only quantify the amount of DNA but also estimate the purity of the DNA sample.
What is a restriction enzyme?
It is an enzyme of bacterial origin that can recognize specific DNA sequences and cut it into segments at those locations.
How is gel electrophoresis used for DNA analysis?
Scientists can load DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size.
Why is genetic profiling important?
DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics. In addition, these advancements recently paved the way for personalized medicine with improved treatment outcomes.
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Popsicle sticks (1/group)5
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Glass funnels (1/group)5
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15 mL conical tubes (2/group)10
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Drinking cups (1/group)5
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1 % NaCl solution (10 mL in a drinking cup/group)50
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Permanent marker (1/group)5
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Beaker for collecting waste (1/group)5
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Lysis buffer I: TE buffer with SDS (TES Buffer) (1 mL/group)5 mL
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Lysis buffer II: TE buffer without SDS (1 mL/group)5 mL
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Proteinase K (10 µL/ group)50 µL
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2.5 M NaCl (100 µL/group)500 µL
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Ethanol 100 % (4°C) (1 mL/group)5 mL
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Glass rods (2/group)10
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Stopwatch (1/group)10
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Microfuge tubes (1.5 mL) (a beaker full/station)1 per station
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Floating foam microfuge racks (1/station)1 per station
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Cell phone with a camera1 per group
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Automated pippete sets (1 µL-1 mL)-1 Dependent on the lab size
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Pipette tips (1 µL-1 mL) 1box each/group-1 Dependent on the lab size
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Microfuge centrifuge-1 Dependent on the lab size
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Water bath (set to 56°C)-1 Dependent on the lab size
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Nuclease free water (125 µL aliquot/group)125 µL
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Lamda DNA (12 µL/group)12 µL
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NEB buffer 2.1 (15 µL/group)15 µL
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Restriction enzyme 1 (StuI) (1 µL/group)1 µL
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Restriction enzyme 2 (BsrGI) (1 µL/group)1 µL
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Stop watch (1/group)5
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Enzyme cooler-1 Dependent on the lab size
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Water bath (set to 37°C)-1 Dependent on the lab size
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Computer with internet connection-1 Dependent on the lab size
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Agarose Powder-1 Dependent on the lab size
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Tris-Acetate-EDTA buffer-1 Dependent on the lab size
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Syber safe gel stain (10,000X) (10 µl/100mL gel)-1 Dependent on the lab size
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6X loading dye-1 Dependent on the lab size
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1 kb DNA Ladder-1 Dependent on the lab size
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Weighing balance1 whole class
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Microwave1 whole class
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DNA gel apparatus1 whole class
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UV transilluminator1 whole class
- 솔루션 및 재료 준비
- 참고: 이 실험실이 원활하게 진행되려면 실험실 클래스당 12개의 샘플을 초과하지 않는 것이 좋습니다.
- 먼저 아이스 캔디 스틱, 깔때기, 15mL 원뿔형 튜브, 마이크로피펫, 얼음이 든 얼음 양동이, 작은 컵 및 미세 원심분리기 튜브를 포함하여 필요한 재료를 수집합니다.
- 다음으로 실험실 실습의 1부에서 사용한 수조를 56°C로 설정합니다.
- 실험실 실습의 두 번째 부분에서 사용할 두 번째 수조를 37°C로 설정합니다.
- TE 버퍼를 만들려면 먼저 400mL의 RO 물을 측정하십시오.
- 그런 다음 1M Tris-HCL pH 8 5 mL를 첨가 한 다음 0.5 M EDTA 용액 1 mL를 첨가합니다.
- 다음으로 용액을 최대 500mL까지 끌어 올리기에 충분한 RO 물을 추가합니다.
- SDS 또는 TES 버퍼로 TE 버퍼를 만들려면 다시 400mL의 RO 물을 측정하십시오.
- 1M Tris-HCL pH 8 5 밀리리터를 첨가 한 다음 0.5 몰 EDTA 용액 1 mL를 첨가합니다.
- 이제 5 mL의 10 % SDS 용액을 추가합니다.
- RO 물을 사용하여 부피를 최대 500mL까지 가져옵니다.
- 다음으로, 학생들이 뺨 세포를 채취하는 데 사용할 1% 식염수를 준비합니다. 깨끗하고 빈 음료수 병에 NaCl 5g을 RO 물 500mL에 녹입니다.
- 학생들이 사용할 수 있도록 작은 음료수 컵에 10mL 분취량을 붓습니다. 학생 그룹당 하나씩 사용합니다.
- 그 후, 학생들이 사용할 수있는 100 % 에탄올 10mL 분취량을 충분히 준비하십시오. 참고: 이러한 에탄올 분취액은 냉동실이나 냉장고에 보관하여 가급적이면 얼음처럼 차갑게 유지하십시오.
Q1: What materials do you need to prepare before starting a DNA isolation lab?
Gather popsicle sticks, funnels, 15 mL conical tubes, micropipettes, ice buckets with ice, small cups, and microcentrifuge tubes. Prepare two water baths set to 56°C and 37°C for different lab stages. Limit the lab to no more than 12 samples per class to ensure smooth execution and proper handling of all materials.
Q2: How do you prepare TE buffer for DNA isolation?
Measure 400 mL of reverse osmosis (RO) water, then add 5 mL of 1M Tris-HCl pH 8 and 1 mL of 0.5 M EDTA solution. Fill the remaining volume to 500 mL with RO water. This buffer protects DNA during isolation and restriction enzyme analysis procedures.
Q3: What is the difference between TE buffer and TES buffer?
TES buffer includes all TE buffer components—400 mL RO water, 5 mL of 1M Tris-HCl pH 8, and 1 mL of 0.5 M EDTA—plus an additional 5 mL of 10% SDS solution. The SDS detergent helps lyse cell membranes during DNA extraction. Both are brought to 500 mL final volume with RO water.
Q4: How should you prepare the saline solution for cheek cell collection?
Dissolve 5 grams of NaCl in 500 mL of reverse osmosis water in a clean soda bottle to create a 1% saline solution. Distribute 10 mL aliquots into small drinking cups, providing one cup per student group for collecting cheek cells during the lab.
Q5: Why is ethanol stored in the freezer or refrigerator before the lab?
Ethanol is stored ice-cold to facilitate DNA precipitation during the isolation process. Prepare 10 mL aliquots of 100% ethanol and keep them refrigerated or frozen. Cold ethanol increases DNA solubility differences, making it precipitate more effectively from the aqueous solution.
Q6: What temperature settings are required for the water baths in this lab?
Set one water bath to 56°C for part one of the laboratory exercise and a second water bath to 37°C for part two. These specific temperatures optimize enzyme activity and cell lysis during different stages of DNA isolation and restriction enzyme analysis.
Q7: How much TE buffer or TES buffer should you prepare for a typical lab class?
Prepare 500 mL of TE buffer or TES buffer as the standard preparation volume. Since the lab accommodates up to 12 samples per class, this volume provides sufficient buffer for cell lysis, DNA extraction, and subsequent washing steps across all student groups.