The following few questions are regarding the steps of a miniprep and restrictio
ID: 225776 • Letter: T
Question
The following few questions are regarding the steps of a miniprep and restriction analysis of plasmid DNA experiment.
a) Why shouldn’t you vortex the sample after adding the lysis buffer during a plasmid prep?
b) Explain the principle of binding and elution of plasmid using a spin column.
c) Why is it important to remove all of the ethanol before eluting the DNA in water or buffer?
d) How does the restriction enzyme digestion of the plasmid inform whether the gene is inserted in the correct orientation?
Much appreciated
Explanation / Answer
a) Why shouldn’t you vortex the sample after adding the lysis buffer during a plasmid prep?
Answer - During Centrifugation , gDNA (bound to protein) forms a pellet while plasmid DNA remains soluble. It is key at this step not to vortex or mix the sample vigorously because gDNA breaks easily, and broken gDNA may be small enough to reanneal and go into solution with the plasmid.
b) Explain the principle of binding and elution of plasmid using a spin column.
Answer -
lysis buffers contain a high concentration of chaotropic salts. Chaotropes have two important roles in nucleic acid extraction. Firstly, they destabilize hydrogen bonds, van der Waals forces and hydrophobic interactions, leading to destabilization of proteins, including nucleases. Secondly, they disrupt the association of nucleic acids with water, thereby providing optimal conditions for their transfer to silica.
Chaotropic salts include guanidine HCL, guanidine thiocyanate, urea, and lithium perchlorate.
In addition to chaotropes, a detergent is often present in the lysis buffer to aid protein solubilization and cell lysis. Enzymes may also feature here, depending on sample type. The broad-spectrum serine protease proteinase K is very efficient in digesting proteins away from nucleic acid preparations. Proteinase K works best under protein denaturing conditions (i.e. in denaturing lysis buffer). Another popular enzyme here, lysozyme, does not work under denaturing conditions and will be most active before the addition of denaturing salts.
Bear in mind that lysis for plasmid isolation is very different to lysis for RNA or genomic DNA extraction because plasmids must be separated from genomic DNA first. The addition of chaotropes will release all types of DNA at once, losing the ability to differentiate small circular DNA from high molecular weight chromosomes. Therefore, in plasmid preps the chaotropes are not added until after cell lysis.
c) Why is it important to remove all of the ethanol before eluting the DNA in water or buffer?
Answer - All ethanol MUST be removed so that the DNA can be successfully removed/eluted from the silica membrane.
d) How does the restriction enzyme digestion of the plasmid inform whether the gene is inserted in the correct orientation?
Answer -
Ideally, you will find two different restriction enzymes for your subcloning. It is also possible to use a single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned in the correct orientation.
If you cannot find enzymes that meet these criteria, do not fear. You have other options, such as:
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