Question

A more refined DNA isolation than the procedure in this experiment features extra steps in which protein and RNA are removed from the sample. Consult books or internet sources to ascertain how protein and RNA are removed; identify the source (book or URL) of your information.

Answer 1

While performing an experiment for preparing a Genomic DNA or Plasmid DNA there is always a concern for the underlying contamination of proteins and RNA in the preparation. These contaminations often reduce the yield and can also turn out to be a liability in the downstream processing for other experiments. Protein contamination and RNA contamination can often be found out by using a spectrophotometer and if obtaining OD at 260/280 for DNA is less than 1.8.

The RNA from the preparation can be removed by incorporating RNase A with the DNA sample at a concentration of 2mg/ml. The incubation of the supernatant (DNA Sample) with this protein(which is a ribonuclease) ensures that all the RNA molecules present in the sample are digested and are thus degraded.

Post-treatment with RNase A a phenol-chloroform extraction step is recommended.

Here, an equal volume of Triss EDTA (TE)-saturated phenol chloroform is mixed with the DNA sample for the extraction of protein contaminants. The solution containing the phenol-chloroform and the DNA is then vortexed vigorously for 30-60 seconds. Centrifugation at the highest setting is then performed to separate out the aqueous and the organic phases. Protein contaminants get into the organic phase whereas the DNA stays in the aqueous phase in the tube.

A user might observe three consecutive phases in the tube after centrifugation is completed, the First phase is the aqueous phase that contains the DNA, the middle phase which will appear to be whitish or hazy is because of the presence of the precipitated protein molecules. The last phase is the organic phase which holds all our protein contaminants. The aqueous phase is carefully removed in a fresh microcentrifuge tube and processed for obtaining pure DNA. It is mandatory not to leave behind some mm of aqueous phase liquid in order to not pipette out the organic phase containing protein.  

So this is how we can get rid of the RNA first, with the help of a ribonuclease called RNase A, which is a protein and might act as a contaminant itself and is thus removed later along with other cellular protein contaminants via phenol-chloroform extraction step. The DNA Sample is then further subjected to the usual alcohol precipitation step as per the general protocol standardized in the laboratory to obtain a pure sample/preparation.   

Reference:

1.) https://www.addgene.org/protocols/purify-plasmid-dna/#phenol

2) http://tools.thermofisher.com/content/sfs/brochures/TN52646-E-0215M-NucleicAcid.pdf

3) http://physiology.med.cornell.edu/faculty/mason/lab/zumbo/files/PHENOL-CHLOROFORM.pdf