Isolating Genomic DNA using PureLink Kits

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Questions and Answers

What is the size of the isolated DNA suitable for downstream applications?

100-200 kb

50-100 kb

20-50 kb (correct)

10-20 kb

What is the purpose of adding RNase A to the sample?

To remove residual RNA from the sample (correct)

To remove impurities from the lysate

To enhance Proteinase K activity

To denature protein

What is the temperature at which the sample is incubated with Proteinase K?

65°C

37°C

45°C

55°C (correct)

What is the purpose of adding ethanol to the lysate?

To mix well with the PureLink Genomic Lysis/Binding Buffer Signup and view all the answers

What is the component that binds to the silica-based membrane?

Genomic DNA Signup and view all the answers

What is the purpose of the PureLink Genomic Wash Buffers?

To remove impurities from the silica-based membrane Signup and view all the answers

What is the volume of the blood sample that can be added to the microcentrifuge tube?

≤200 μL Signup and view all the answers

What is the step that follows the addition of ethanol to the lysate?

Binding DNA to the silica-based membrane Signup and view all the answers

What is the optimal temperature for long-term storage of purified DNA?

-20°C Signup and view all the answers

What is the primary purpose of restriction endonucleases in recombinant DNA technology?

To specifically cut DNA into manageable fragments Signup and view all the answers

What is the composition of 5X TBE solution for electrophoresis?

54 g Tris base + 27.5 g Boric Acid and 20 ml of 0.5 M EDTA Signup and view all the answers

Why is it recommended to avoid repeated freezing and thawing of DNA?

To prevent DNA degradation Signup and view all the answers

What is the typical length of recognition sites for restriction endonucleases?

6 to 8 bp Signup and view all the answers

What is the primary purpose of DNA electrophoresis?

To separate DNA fragments based on size Signup and view all the answers

Why is Ethidium bromide used in DNA electrophoresis?

To visualize DNA fragments Signup and view all the answers

What is the purpose of using a water bath at 37°C in DNA restriction digestion?

To digest DNA with restriction enzymes Signup and view all the answers

What happens to the DNA molecules during gel electrophoresis?

They migrate to a distance inversely proportional to their molecular weight Signup and view all the answers

What is the purpose of incubating the DNA sample with the restriction enzyme?

To digest the DNA molecules at specific recognition sites Signup and view all the answers

Why do longer DNA molecules move more slowly during gel electrophoresis?

Because they are impeded more by the agarose matrix Signup and view all the answers

What is the purpose of ethidium bromide in gel electrophoresis?

To visualize DNA bands under UV light Signup and view all the answers

What is the recommended temperature for the restriction enzyme digestion reaction?

37ºC Signup and view all the answers

What is the function of the agarose gel in gel electrophoresis?

To separate DNA molecules based on their size and shape Signup and view all the answers

Why is it important to wear gloves and eye protectors when working with ethidium bromide and UV light?

To prevent exposure to mutagenic and carcinogenic substances Signup and view all the answers

What is the direction of DNA migration in an electric field?

From the negative pole to the positive pole Signup and view all the answers

What determines the density of the agarose matrix in gel electrophoresis?

The concentration of the agarose Signup and view all the answers

What is the thickness of the agarose gel typically used in gel electrophoresis?

5 mm Signup and view all the answers

What is the unit of enzyme defined as?

The amount of enzyme needed to completely digest 1 microgram of DNA in 1 hour Signup and view all the answers

What is the purpose of adding the loading buffer to the digested sample?

To visualize the DNA bands in the agarose gel Signup and view all the answers

What is the purpose of running the non-digested sample on the agarose gel?

To compare the digested and non-digested samples Signup and view all the answers

What is the composition of the agarose gel?

A linear polymer derived from seaweed Signup and view all the answers

What is the purpose of the PureLink Genomic DNA Kits?

To efficiently isolate genomic DNA from mammalian cells and tissues Signup and view all the answers

What is the unit of measurement for the micropipettes used in the lab?

μl Signup and view all the answers

What is the formula used to calculate dilutions?

C1V1 = C2V2 Signup and view all the answers

What is the purpose of the weighing trays in the lab?

To weigh out chemicals for solution preparation Signup and view all the answers

What is the term for the concentration of a solution in grams per liter?

wt/vol Signup and view all the answers

What is the purpose of the hot plate in the lab?

To heat up solutions Signup and view all the answers

What is the term for the concentration of a solution in parts per million?

ppm Signup and view all the answers

What is the purpose of the graduated cylinders in the lab?

To measure the volume of solutions accurately Signup and view all the answers

Study Notes

DNA Extraction and Purification

The PureLink Genomic DNA Kits allow for rapid and efficient purification of genomic DNA from various sources, including blood samples.
The kit is designed to isolate genomic DNA from mammalian cells and tissues, such as mouse/rat tail, blood samples, buccal swabs, bacteria, and yeast.
The extracted DNA is suitable for various downstream applications, including PCR, restriction enzyme digestion, and Southern blotting.
The isolated DNA is 20–50 kb in size.

Preparing Lysates

To prepare a lysate, a fresh or frozen blood sample is added to a sterile microcentrifuge tube.
Proteinase K and RNase A are then added to the sample, followed by incubation at room temperature for 2 minutes.
The sample is then incubated at 55°C for 10 minutes to promote protein digestion.
Ethanol is added to the lysate, and the mixture is vortexed for 5 seconds to yield a homogenous solution.

Binding DNA

The lysate is mixed with PureLink Genomic Lysis/Binding Buffer and ethanol, and the mixture is vortexed for 5 seconds.
The resulting solution is then bound to a silica-based membrane in a column or plate.
Impurities are removed by thorough washing with Wash Buffers.
The genomic DNA is then eluted in low salt Elution Buffer.

DNA Restriction (Digestion)

Restriction enzymes are used to specifically cut long pieces of genomic DNA into manageable fragments.
Each restriction enzyme has its own specific recognition site on double-stranded DNA, usually 6 to 8 bp in length and palindromic in sequence.
Typical digestions include a unit of enzyme per microgram of starting DNA, and one enzyme unit usually is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature.

Gel Electrophoresis

Gel electrophoresis is a powerful technique in molecular biology that separates molecules based on their charge, size, and other physical features.
DNA molecules are negatively charged on their phosphate groups and travel from the negative pole to the positive pole in an electric field.
The gel functions as a sieve through which DNA migrates to a distance inversely proportional to its molecular weight.
The resulting bands can be visualized using ethidium bromide staining, which intercalates in the DNA double helix and gives a fluorescent glow upon exposure to UV.

Laboratory Techniques and Precautions

Various laboratory techniques are used in molecular biology, including PCR, restriction enzyme digestion, and Southern blotting.
General techniques and precautions include solution preparation, DNA extraction and electrophoresis, DNA digestion and electrophoresis, competent cell preparation, bacterial transformation, molecular cloning, and screening for recombinants.
Personal protective equipment, such as gloves and eye protectors, should be worn when handling mutagenic and carcinogenic materials like ethidium bromide and UV.

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Description

This quiz covers the process of isolating genomic DNA using PureLink Genomic DNA Kits, including the selective binding of DNA to silica-based membrane and digestion with Proteinase K.