Purification of plasmid DNA Flashcards
What are plasmids?
Self-replicating circles of double stranded DNA found in many bacterial cells.
Why is purification of plasmid more difficult than purification of total bacterial DNA?
The plasmid DNA must be separated from the bacterial chromosomal DNA which is more difficult.
What are some physical differences between plasmid DNA and bacterial DNA?
Plasmid DNA is much smaller than bacterial DNA and plasmid DNA remains circular instead of forming linear fragments during preparation of the cell extract when broken down.
What is one way to separate the plasmid DNA from the bacterial DNA?
The cells can be lysed in an isotonic solution (so minimal damage to chromosomal DNA occurs) and a detergent (Triton X-100) can be used to rupture the membrane. The chromosomal DNA remains in large pieces and co-pellets with the cell debris upon centrifugation, whereas the plasmids remain in solution which can be pipetted to a fresh tube.
What steps can be taken to further purify the plasmid DNA?
Ehtidium bromide-caesium chloride density gradient centrifugation can be used to remove residual chromosomal DNA is ultra pure plasmid DNA is needed.
How does ethidium bromide-caesium chloride work to purify DNA?
Ethidium bromide intercalates between the strands of the double helix to reduce the buoyant density. The density of linear DNA is reduced more than supercoiled DNA (undamaged plasmids) which can allow the two to be separated on a caesium-bromide gradient.
What happens after ethidium bromide has been added to the solution?
The solution is mixed with caesium chloride and is centrifuged at a high speed for at least 24 hours. A density gradient is produced Different bands will be formed in the gradient which will depend on the buoyant density.
Where will the different components in the ethidium bromide-caesium chloride density centrifugation form?
DNA will migrate to around 1.7g cm-3 (middle of tube), protein will rise to the top of the tube (lower buoyant density) and RNA will form pellets at the bottom.
How can the plasmid DNA be removed from the chromosomal DNA?
As the linear and supercoiled DNA will band separately (linear above the hypercoiled) the plasmid DNA can be removed with a hypodermic syringe.
How can contaminant EtBr and CsCl be removed from the plasmid sample after centrifugation?
EtBr can be removed with butanol and CsCl can be removed by dialysis.
What is a useful tag on mRNA that allows it to be separated from the rest of RNA?
mRNA is polyadenylated at the 3’ end (string of adenines attached) which acts as a useful tag to select the mRNA from the rest of the RNA.
What is the method used to separate the mRNA?
Oligo(dT) chromatography.
What is the first step in the basic method for Oligo(dT) chromatography?
Firstly, prepare a small column containing Oligo(dT) bound to a cellulose matrix.
What happens in the second stage of Oligo(dT) chromatography?
The total RNA is added in a high salt buffer and the Poly(A) tails of the mRNA will bind to the oligo-dT by hydrogen bonding.
What is the third step in oligo(dT) chromatography?
A medium salt buffer can be added that washes of rRNA and tRNA - these are not bound to the oligo-dT.
What is added after the third step in oligo-dT chromatography?
A buffer without salt is then added to the column to wash off the polyA RNA that has been stuck to the oligo-dt.
How can the RNA then be concentrated in the final step of oligo-dT chromatography?
Ethanol precipitation can be used to concentrate the RNA.
What is a variation of oligo-dT chromatography?
The oligo-dT can be attached to magnetic beads and a magnet can be used to isolate the mRNA, as everything else is washed off. The mRNA will be stuck to the magnet.
How does purification of total eukaryotic DNA differ from the purification of DNA from bacteria?
A convenient material will be chosen such as young leaves, liver cells, the material is usually mechanically disrupted such as grinding it in liquid nitrogen or sonication and extra steps may be required and materials may be challenging and require special procedures.
What are some examples of enzymes that are used to manipulate nucleic acids?
Nucleases, ligases, polymerases and modifying enzymes.
What are the functions of each of the different types of enzymes used to manipulate nucleic acids?
Nucleases cut, shorten or degrade nucleic acids, ligases join molecules together, polymerases make copies of nucleic acid molecules and mofiying enzymes remove or add chemical groups.
What are the two subclasses of nucleases?
Exonucleaes that remove nucleotides one at a time from the ends of a nucleic acid molecule and endonucleases that are able to break internal phosphodiester bonds within a nucleic acid molecule.
Give two examples of one type of nuclease.
Bal31 is an exonuclease that digests the ends of DNA and can be used to shorten DNA molecules. The degree of digestion is dependent on the time and concentration. Exonuclease III removes 3’ termini of DNA molecules and leaves single stranded ends or whole molecules, dependent on the conditions of the reaction.
Give two examples of the other type of nuclease.
S1 is an endonuclease that digests only single-stranded DNA, leaving double stranded DNA in tact. It can be used to remove single stranded ends prior to cloning or to differentiate between single and double stranded DNA. Deoxyribonuclease I (DNase I) chops up all DNA and is useful to get rid of DNA or to produce small fragments.