Molecular Biology: DNA Isolation and Purification

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Questions and Answers

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

To protect the DNA from degradation

To separate DNA molecules based on their charge

To act as a sieve, allowing DNA to migrate according to its molecular weight (correct)

To provide a surface for DNA to bind to

What is the purpose of using ethidium bromide in gel electrophoresis?

To increase the resolution of the gel

To visualize DNA bands by intercalating into the DNA double helix (correct)

To prevent DNA degradation during the process

To enhance the migration of DNA molecules

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

To enhance the visualization of DNA bands

To protect against the mutagenic and carcinogenic properties of ethidium bromide and UV light (correct)

To prevent contamination of the sample

To improve the resolution of the gel

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 purpose of casting the agarose gel in a mold with a comb?

To create separate wells for loading the DNA samples Signup and view all the answers

How can the time required for the agarose gel to harden be shortened?

By pouring the melted agarose into the refrigerator Signup and view all the answers

What is the recommended temperature for centrifuging the column at 10,000 × g for 1 minute?

room temperature Signup and view all the answers

What is the purpose of adding Wash Buffer 1 prepared with ethanol to the column?

To wash and remove impurities from the DNA Signup and view all the answers

How many times should the column be centrifuged at maximum speed for 1 minute at room temperature during the elution step?

What is the recommended volume of PureLink® Genomic Elution Buffer to add to the column for elution?

25-200 μL Signup and view all the answers

What is the purpose of incubating the column at room temperature for 1 minute during the elution step?

To allow the elution buffer to interact with the DNA Signup and view all the answers

What should be done with the spin column after the second elution step?

Remove and discard it Signup and view all the answers

What is the recommended temperature for storing the purified DNA?

-20°C Signup and view all the answers

Why is it necessary to perform multiple loading of the lysate to the same PureLink® Spin Column?

To process >200 μL starting material Signup and view all the answers

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

20-50 kb Signup and view all the answers

What is the purpose of adding RNase A to the sample during the extraction procedure?

To remove residual RNA from the sample Signup and view all the answers

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

55°C Signup and view all the answers

What is the purpose of the PureLink Genomic Lysis/Binding Buffer?

To aid in protein denaturation and binding to the silica membrane Signup and view all the answers

What is the ratio of sample to Proteinase K added to the microcentrifuge tube?

≤200 μL:20 μL Signup and view all the answers

What is the purpose of the ethanol added to the lysate?

To aid in binding to the silica membrane Signup and view all the answers

What is the final step after adding the PureLink Genomic Binding Buffer to the sample?

Proceeding immediately to Binding DNA Signup and view all the answers

What is the purpose of the PureLink Genomic Wash Buffers?

To remove impurities from the bound DNA Signup and view all the answers

What is the purpose of adding EDTA to the TBE buffer?

To provide a chelating agent for metal ions Signup and view all the answers

What is the optimal concentration of agarose for separating linear DNA molecules of 0.5 to 7 kb?

0.9% Signup and view all the answers

Why is it recommended to use a voltage of 1-5 V/cm for agarose gel electrophoresis?

To prevent the heating of the gel Signup and view all the answers

What is the primary function of the bromophenol blue in the gel-loading buffer?

To provide a tracking dye for DNA migration Signup and view all the answers

What is the purpose of soaking the gel in ethidium bromide after migration?

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

What is the concentration of EDTA in the 5X TBE buffer?

0.5 M Signup and view all the answers

Why is it important to handle ethidium bromide with care?

It is a carcinogen and toxic Signup and view all the answers

What is the purpose of using a micropipette to load the DNA samples into the gel?

To slowly and carefully load the DNA into the gel Signup and view all the answers

What would be the impact of contamination with protein or phenol on the DNA concentration measurement?

The DNA concentration measurement would be significantly lower than the actual value Signup and view all the answers

What is the purpose of diluting the DNA sample in the procedure?

To reduce the effect of contamination on the DNA concentration measurement Signup and view all the answers

What is the formula to calculate the DNA concentration from the OD260 reading?

OD260 * 50 ng/µl * dilution factor Signup and view all the answers

What is the temperature of the water bath and incubator used in the competent cell preparation?

37°C Signup and view all the answers

What is the purpose of the calcium chloride solution in the competent cell preparation?

To make the E. coli cells competent for transformation Signup and view all the answers

What is the final storage condition of the competent cells?

At -80°C Signup and view all the answers

What is the purpose of the preincubation step at 37°C for 1 hour in the competent cell preparation?

To allow the E. coli cells to grow and multiply Signup and view all the answers

What is the composition of the 100 ml Ice CaCl2 solution used in the competent cell preparation?

60 mM CaCl2, 15% glycerol, 10 mM PIPES, pH 7.0 Signup and view all the answers

Study Notes

DNA Extraction and Purification

The PureLink Genomic DNA Kits enable the extraction and purification of DNA from various starting materials, including tissues, cells, or blood.
The extracted DNA is suitable for downstream applications such as PCR, restriction enzyme digestion, and Southern blotting.
The DNA binds to a silica-based membrane, and impurities are removed by washing with Wash Buffers.
The genomic DNA is then eluted in low salt Elution Buffer.

Extraction Procedure

The extraction procedure involves preparing lysates, binding DNA, washing DNA, and eluting DNA.
The lysate is prepared by adding Proteinase K and RNase A to the sample and incubating at 55°C.
The lysate is then mixed with ethanol and PureLink Genomic Lysis/Binding Buffer.
The DNA binds to the silica-based membrane, and the impurities are removed by washing with Wash Buffers.
The genomic DNA is then eluted in low salt Elution Buffer.

DNA Isolation and Purification

The isolated DNA is 20-50 kb in size.
The DNA is suitable for various downstream applications.
The extracted DNA is eluted in low salt Elution Buffer.

Gel Electrophoresis

Gel electrophoresis is a technique used to separate DNA molecules based on their size.
The gel functions as a sieve, allowing DNA molecules to migrate to a distance inversely proportional to their molecular weight.
Ethidium bromide is used to stain the DNA, allowing it to be visualized under UV light.
Attention must be paid to the handling of ethidium bromide, as it is mutagenic and carcinogenic.

Agarose Gel Electrophoresis

Agarose gels are cast by melting agarose in the presence of a buffer.
The melted solution is poured into a mold and allowed to harden at room temperature.
The agarose forms a matrix, and the density of the matrix is determined by the concentration of agarose.
The gel is typically 5 mm thick.

DNA Migration

DNA migration occurs in an electrical field.
The working solution is typically 1X dilution, but 0.5X provides more than enough buffering power.
The samples are mixed with a loading buffer and loaded into the slots of the submerged gel.

DNA Visualization

Ethidium bromide is added to the soaking buffer to a final concentration of 0.5 µg/ml.
The gel is examined under UV illumination, and the DNA is visualized.

DNA Quantitation

DNA quantitation is performed using a spectrophotometer.
The DNA sample is diluted, and the DNA concentration is calculated using the formula: OD260 * 50 ng/µl * dilution factor.

Competent Cell Preparation

Competent cells are prepared using the calcium chloride method.
A glycerol cell culture stock of the respective E. coli strain is thawed and added to liquid medium.
The culture is preincubated at 37°C for 1 hour, then transferred to an incubator-shaker for 2-3 hours.
The cells are pelleted by centrifugation, resuspended in calcium chloride solution, and incubated in an ice-water bath.
The resulting cell pellet is re-suspended in ice-cold calcium chloride solution to yield the final competent cell suspension.
Competent cells are stored at -80°C.

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Description

This quiz covers the process of isolating and purifying genomic DNA using PureLink Genomic DNA Kits. Topics include DNA binding to silica-based membrane, lysate preparation, and digestion with Proteinase K.