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Questions and Answers
These enzymes break polypeptides down into smaller units, which are more easily removed by ______.
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.
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 ______.
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.
One of these methods is ______-exchange chromatography, which separates molecules according to how tightly they bind to electrically charged particles.
DNA and RNA are both negatively charged, as are some proteins, and so bind to a positively charged ______.
DNA and RNA are both negatively charged, as are some proteins, and so bind to a positively charged ______.
The extract passes through the column, and because this extract contains very little ______, all the negatively charged molecules bind to the resin.
The extract passes through the column, and because this extract contains very little ______, all the negatively charged molecules bind to the resin.
By gradually increasing the salt concentration, different types of molecule can be ______ from the resin one after another.
By gradually increasing the salt concentration, different types of molecule can be ______ from the resin one after another.
The most frequently used method of concentration is ______ precipitation.
The most frequently used method of concentration is ______ precipitation.
DNA concentrations can be accurately measured by ultraviolet (UV) absorbance ______.
DNA concentrations can be accurately measured by ultraviolet (UV) absorbance ______.
The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of ______ in the sample.
The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of ______ in the sample.
Absorbance is usually measured at ______ nm, with an absorbance of 1.0 corresponding to 50 mg of double-stranded DNA per ml.
Absorbance is usually measured at ______ nm, with an absorbance of 1.0 corresponding to 50 mg of double-stranded DNA per ml.
A pure sample of DNA has an A260/A280 ratio of ______.
A pure sample of DNA has an A260/A280 ratio of ______.
If the A260/A280 ratio is less than 1.8, it indicates contamination with either ______ or phenol.
If the A260/A280 ratio is less than 1.8, it indicates contamination with either ______ or phenol.
Total cell DNA from ______ or animals will be needed if cloning genes from these organisms.
Total cell DNA from ______ or animals will be needed if cloning genes from these organisms.
Growth of cells in liquid medium is appropriate only for ______, other microorganisms, and plant and animal cell cultures.
Growth of cells in liquid medium is appropriate only for ______, other microorganisms, and plant and animal cell cultures.
Ethanol is used to collect precipitated DNA by ______.
Ethanol is used to collect precipitated DNA by ______.
Supercoiled molecules form a band in an EtBr–CsCl gradient at a different position to ______ and open-circular DNA.
Supercoiled molecules form a band in an EtBr–CsCl gradient at a different position to ______ and open-circular DNA.
Ethidium bromide–caesium chloride density gradient centrifugation is a very efficient method for obtaining pure ______ DNA.
Ethidium bromide–caesium chloride density gradient centrifugation is a very efficient method for obtaining pure ______ DNA.
When a cleared lysate is subjected to density gradient centrifugation, plasmids band at a distinct ______.
When a cleared lysate is subjected to density gradient centrifugation, plasmids band at a distinct ______.
Partial unwinding of the DNA double helix is caused by EtBr intercalation between adjacent ______.
Partial unwinding of the DNA double helix is caused by EtBr intercalation between adjacent ______.
The normal DNA molecule is partially unwound by taking up four ______ molecules.
The normal DNA molecule is partially unwound by taking up four ______ molecules.
In the purification process, supercoiled DNA is removed with a ______.
In the purification process, supercoiled DNA is removed with a ______.
EtBr–CsCl density gradient centrifugation involves the separation of DNA into different ______.
EtBr–CsCl density gradient centrifugation involves the separation of DNA into different ______.
To extract EtBr from the solution, n-butanol is used to allow layers to ______.
To extract EtBr from the solution, n-butanol is used to allow layers to ______.
CsCl diffuses into the ______ during the purification process.
CsCl diffuses into the ______ during the purification process.
After extraction of the EtBr, the final step is the removal of ______ by dialysis.
After extraction of the EtBr, the final step is the removal of ______ by dialysis.
Plasmid amplification offers a means of increasing the ______ of a plasmid.
Plasmid amplification offers a means of increasing the ______ of a plasmid.
Some multi-copy plasmids have the useful property of being able to replicate in the absence of ______ synthesis.
Some multi-copy plasmids have the useful property of being able to replicate in the absence of ______ synthesis.
During plasmid amplification, an inhibitor of protein synthesis, such as ______, is added to the culture.
During plasmid amplification, an inhibitor of protein synthesis, such as ______, is added to the culture.
After adding chloramphenicol, the culture is incubated for a further ______ hours.
After adding chloramphenicol, the culture is incubated for a further ______ hours.
The resulting plasmid preparation is virtually ______% pure and ready for use as a cloning vector.
The resulting plasmid preparation is virtually ______% pure and ready for use as a cloning vector.
Plasmids make up only a small proportion of the total DNA in the ______ cell.
Plasmids make up only a small proportion of the total DNA in the ______ cell.
The amplification process can achieve plasmid copy numbers of several ______.
The amplification process can achieve plasmid copy numbers of several ______.
The key difference between phage DNA purification and plasmid DNA preparation is that for phages the starting material is not normally a ______ extract.
The key difference between phage DNA purification and plasmid DNA preparation is that for phages the starting material is not normally a ______ extract.
The term conformation refers to the overall spatial configuration of the molecule, with the two simplest conformations being linear and ______.
The term conformation refers to the overall spatial configuration of the molecule, with the two simplest conformations being linear and ______.
Plasmids and the bacterial chromosome are ______ in shape.
Plasmids and the bacterial chromosome are ______ in shape.
During cell extract preparation, the chromosome is always broken to give ______ fragments.
During cell extract preparation, the chromosome is always broken to give ______ fragments.
A method for separating circular from ______ molecules will result in pure plasmids.
A method for separating circular from ______ molecules will result in pure plasmids.
The usual stage at which size fractionation is performed is during preparation of the cell ______.
The usual stage at which size fractionation is performed is during preparation of the cell ______.
Controlled lysis is performed to prevent ______ of the bacterial DNA.
Controlled lysis is performed to prevent ______ of the bacterial DNA.
Treatment with EDTA and lysozyme is carried out in the presence of ______, preventing the cells from bursting immediately.
Treatment with EDTA and lysozyme is carried out in the presence of ______, preventing the cells from bursting immediately.
Adding a non-ionic detergent such as ______ induces cell lysis while causing very little breakage of bacterial DNA.
Adding a non-ionic detergent such as ______ induces cell lysis while causing very little breakage of bacterial DNA.
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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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