Experiment 2 Report - Got 10/10. PDF

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Got 10/10....

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Jackson Prior

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Experiment 2 Report Questions 1. Lines by letter: a. Chromosomal DNA b. 23s rRNA c. 16S rRNA d. 5s rRNA + tRNA

2. One function is to act with chloroform as a lysing agent to precipitate other contaminants (chromosomal DNA, proteins and lipids) into an insoluble mass, isolating the RNA. It also deactivates ribonucleases in cell lysing buffer, preventing breakdown of RNA.

3. mRNA is visible as a ‘smear’ seen on either side of, and between, B and C. mRNAs are heterologous, meaning not uniform in length. Hence, they will all travel different distances, being present over the gel in different bands, creating a blurred or smeared effect.

4. The purpose of heating the sample to 90oC is to break the H-bonds in the RNA, destroying the double-stranded linkages and forcing a single-stranded conformation. The RNAses are deactivated by the phenol/chloroform mixture prior to the heating, which is maintained by the guanidinium isothiocyanate in the gel. Hence the only correct answer is RNA denaturation.

5. It would look no different. The purpose of heating and denaturation is to break the hydrogen bonding between base pairs of the tRNA and rRNA to conform it into a single-stranded structure. mRNA is naturally occurring as a single-stranded molecule; thus the removal of these denaturing aspects will not change the outcome of the mRNA structure and migration through the gel.

6. Heating to 90OC breaks hydrogen bonds between the base pairs of RNA, causing the initial denaturing from a double stranded to a single stranded molecule. When it enters the gel, the denaturants present in the FFMB dye maintain the denatured state of RNA by preventing the H-bonds from reforming.

7. RNA is already denatured once it is placed into the cast (after being exposed to 90 oC heating). The 5 mm guanidinium isothiocyanate is simply there to maintain this denatured state - but does not cause it.

8. PLG is able to separate the polar and non-polar phases of phenol/chloroform cell lysate as it has an intermediate density between the organic and aqueous phases. After centrifugation, the less dense (aqueous phase) layer sits above PLG, whilst the more dense (organic phase) falls beneath the PLG....