DNA Purification Techniques
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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

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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.

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