DNA Purification Techniques
42 Questions
0 Views

DNA Purification Techniques

Created by
@InnovativeWilliamsite5857

Podcast Beta

Play an AI-generated podcast conversation about this lesson

Questions and Answers

These enzymes break polypeptides down into smaller units, which are more easily removed by ______.

phenol

The only effective way to remove the RNA is with the enzyme ______, which rapidly degrades these molecules into ribonucleotide subunits.

ribonuclease

Biochemists have devised various methods for using differences in electrical charge to separate mixtures of chemicals into their individual ______.

components

One of these methods is ______-exchange chromatography, which separates molecules according to how tightly they bind to electrically charged particles.

<p>ion</p> Signup and view all the answers

DNA and RNA are both negatively charged, as are some proteins, and so bind to a positively charged ______.

<p>resin</p> Signup and view all the answers

The extract passes through the column, and because this extract contains very little ______, all the negatively charged molecules bind to the resin.

<p>salt</p> Signup and view all the answers

By gradually increasing the salt concentration, different types of molecule can be ______ from the resin one after another.

<p>detached</p> Signup and view all the answers

The most frequently used method of concentration is ______ precipitation.

<p>ethanol</p> Signup and view all the answers

DNA concentrations can be accurately measured by ultraviolet (UV) absorbance ______.

<p>spectrophotometry</p> Signup and view all the answers

The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of ______ in the sample.

<p>DNA</p> Signup and view all the answers

Absorbance is usually measured at ______ nm, with an absorbance of 1.0 corresponding to 50 mg of double-stranded DNA per ml.

<p>260</p> Signup and view all the answers

A pure sample of DNA has an A260/A280 ratio of ______.

<p>1.8</p> Signup and view all the answers

If the A260/A280 ratio is less than 1.8, it indicates contamination with either ______ or phenol.

<p>protein</p> Signup and view all the answers

Total cell DNA from ______ or animals will be needed if cloning genes from these organisms.

<p>plants</p> Signup and view all the answers

Growth of cells in liquid medium is appropriate only for ______, other microorganisms, and plant and animal cell cultures.

<p>bacteria</p> Signup and view all the answers

Ethanol is used to collect precipitated DNA by ______.

<p>centrifugation</p> Signup and view all the answers

Supercoiled molecules form a band in an EtBr–CsCl gradient at a different position to ______ and open-circular DNA.

<p>linear DNA</p> Signup and view all the answers

Ethidium bromide–caesium chloride density gradient centrifugation is a very efficient method for obtaining pure ______ DNA.

<p>plasmid</p> Signup and view all the answers

When a cleared lysate is subjected to density gradient centrifugation, plasmids band at a distinct ______.

<p>point</p> Signup and view all the answers

Partial unwinding of the DNA double helix is caused by EtBr intercalation between adjacent ______.

<p>base pairs</p> Signup and view all the answers

The normal DNA molecule is partially unwound by taking up four ______ molecules.

<p>EtBr</p> Signup and view all the answers

In the purification process, supercoiled DNA is removed with a ______.

<p>syringe</p> Signup and view all the answers

EtBr–CsCl density gradient centrifugation involves the separation of DNA into different ______.

<p>layers</p> Signup and view all the answers

To extract EtBr from the solution, n-butanol is used to allow layers to ______.

<p>separate</p> Signup and view all the answers

CsCl diffuses into the ______ during the purification process.

<p>buffer</p> Signup and view all the answers

After extraction of the EtBr, the final step is the removal of ______ by dialysis.

<p>CsCl</p> Signup and view all the answers

Plasmid amplification offers a means of increasing the ______ of a plasmid.

<p>yield</p> Signup and view all the answers

Some multi-copy plasmids have the useful property of being able to replicate in the absence of ______ synthesis.

<p>protein</p> Signup and view all the answers

During plasmid amplification, an inhibitor of protein synthesis, such as ______, is added to the culture.

<p>chloramphenicol</p> Signup and view all the answers

After adding chloramphenicol, the culture is incubated for a further ______ hours.

<p>12</p> Signup and view all the answers

The resulting plasmid preparation is virtually ______% pure and ready for use as a cloning vector.

<p>100</p> Signup and view all the answers

Plasmids make up only a small proportion of the total DNA in the ______ cell.

<p>bacterial</p> Signup and view all the answers

The amplification process can achieve plasmid copy numbers of several ______.

<p>thousand</p> Signup and view all the answers

The key difference between phage DNA purification and plasmid DNA preparation is that for phages the starting material is not normally a ______ extract.

<p>cell</p> Signup and view all the answers

The term conformation refers to the overall spatial configuration of the molecule, with the two simplest conformations being linear and ______.

<p>circular</p> Signup and view all the answers

Plasmids and the bacterial chromosome are ______ in shape.

<p>circular</p> Signup and view all the answers

During cell extract preparation, the chromosome is always broken to give ______ fragments.

<p>linear</p> Signup and view all the answers

A method for separating circular from ______ molecules will result in pure plasmids.

<p>linear</p> Signup and view all the answers

The usual stage at which size fractionation is performed is during preparation of the cell ______.

<p>extract</p> Signup and view all the answers

Controlled lysis is performed to prevent ______ of the bacterial DNA.

<p>breakage</p> Signup and view all the answers

Treatment with EDTA and lysozyme is carried out in the presence of ______, preventing the cells from bursting immediately.

<p>sucrose</p> Signup and view all the answers

Adding a non-ionic detergent such as ______ induces cell lysis while causing very little breakage of bacterial DNA.

<p>Triton X-100</p> Signup and view all the answers

Study Notes

Purification of DNA from Living Cells

  • Enzymes: Ribonuclease degrades RNA molecules into ribonucleotide subunits
  • Ion-exchange Chromatography: Separates molecules by their binding ability to electrically charged particles in a resin
    • DNA and RNA are negatively charged and bind to positively charged resins
    • Salt disrupts the electrical attachment, with higher concentrations required to remove more tightly bound molecules
  • Concentration: DNA concentrations can be measured by UV absorbance spectrophotometry
    • Absorbance at 260 nm is directly proportional to the amount of DNA
    • A260 of 1.0 corresponds to 50mg of double-stranded DNA per ml
    • Ratio of A260/A280 indicates purity (1.8 for pure DNA)

Preparation of Plasmid DNA

  • Size Fractionation: Separation of DNA based on size
    • Large DNA fragments are removed by centrifugation
    • Bacterial chromosomes are physically attached to the cell envelope, aiding in their removal
  • Controlled Lysis: Sphaeroplasts (cells with partially degraded cell walls) are formed to prevent chromosomal breakage
    • Addition of non-ionic detergents, like Triton X-100, lyses cells gently
  • EtBr–CsCl Density Gradient Centrifugation: Separates circular plasmids from linear bacterial DNA
    • Plasmids band at a distinct point in the gradient
    • EtBr binds to DNA and fluoresces under UV light
    • EtBr is extracted with n-butanol and CsCl is removed by dialysis

Plasmid Amplification

  • Increased Copy Number: Amplification increases the number of copies of a plasmid in a cell
    • Multicopy plasmids can replicate in the absence of protein synthesis
    • Inhibition of protein synthesis (e.g., with chloramphenicol) allows plasmid replication to continue
  • High Yield: Plasmid copy numbers can reach several thousand, increasing the yield of plasmid DNA

Preparation of Bacteriophage DNA

  • Different Starting Material: Bacteriophage DNA purification begins with phage particles, not a cell extract

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Quiz Team

Related Documents

Description

This quiz explores the various methods used for the purification of DNA from living cells, including enzyme activity, ion-exchange chromatography, and concentration measurement through UV absorbance. Additionally, it covers the preparation and size fractionation of plasmid DNA, outlining key principles and techniques involved in these processes.

More Like This

DNA Purification Process Quiz
10 questions
Molecular Biology: DNA Isolation and Purification
38 questions
Use Quizgecko on...
Browser
Browser