Derek Wong
Biology 275L-03
Lab 11
Total Isolation of RNA of Rat A-10 Cells and E. Coli.
In previous labs, DNA was isolated in this lab RNA will be isolated. RNA differs from DNA in that the base Thymine is replaced by Uracil and the ribose is not deoxyribbose (like how it is in DNA). RNA is also a single stranded molecule, which is then translated to make proteins. These differences make the process to isolate RNA slightly different from DNA. The first is that DNA needs to be removed instead of RNA. The quality and quantity of the RNA is different in that an absorbance of 1 at 260nm corresponds to 40ug/ml. The ratio of 260/280 to 1.8 means that the RNA isolation was relatively pure. The third difference is that there must be RNase-free condition, otherwise the RNA sample will be dissolved before the extraction process will be complete.
There are three different types of RNA, mRNA, tRNA, and rRNA. mRNA contains information about amino acid sequence making polypeptides, tRNA are small pieces of RNA that bind amino acids and directs them to the growing polypeptide chain, and rRNA makes up ribosomes which are the site of protein synthesis.
In this lab, RNA isolation will take place from Rat A-10 cells, and E. Coli. This will take under native conditions, instead of denaturing conditions (which would have help prevented smearing) because formaldehyde is a carcinogen.
Results
Table 1: Spectrophotometry Reults.
|
|
260 nm Abs |
280 nm Abs |
Ratio 260/280 |
Est. [RNA] |
|
E. Coli |
0.196 |
0.170 |
1.1512 |
196 ug/ml |
|
Rat A-10 Cells |
0.136 |
0.122 |
1.1170 |
136 ug/m; |
This table shows the numerical values obtained when the two samples were ran through a spectrophotometer at 260nm and 280nm, the ratio of 260/280m, and the estimated RNA concentration.
Figure 1: Agarose Gel Electrophoresis of RNA isolation of E. Coli and Rat A-10 cells. This depicts the gel electrophoresis of the class RNA isolation of the two samples. Our samples were in Lane 4(Rat A-10 cells) and Lane 5 (E. Coli). Lane 1:DNA Maker (top to bottom, bp = base pairs): 100,000 bp; 80,000 bp; 60,000 bp; 50,000 bp; 40,000 bp; 3, 000 bp; 2, 500 bp; 2,000 bp; 1,500 bp; 1, 000 bp; 750 bp; 500 bp; 250bp.
Discussion
In this lab we isolated RNA from E. Coli and Rat A-10 cells. We used native conditions and took precautions such as having an RNase free-conditions to ensure that the RNA samples will not denature before isolation was complete.
The spectrophotometry readings are indicative samples having a relatively unpure state. This was determined by comparing each ratio of 260/280 to 1.8. The closer the ratio found is to 1.8, then the more pure the sample is. The low ratio could be because of contamination or that some of the RNA was lost in the isolation and extraction process.
Lane 4 (Rat Cells) of the gel shows no band possibly
indicating no RNA present. This could
mean that the RNA denatured and that there was nothing to be ran
on the gel, or it could mean that the concentration of RNA was so minute that
it is not visible. It could be supported that there was not enough RNA in the
sample ran since the estimated concentration of RNA was so minute. Lane 5 (E. Coli)
showed weak bands when compared to the other groups. This could mean again that
the RNA concentration is low causing weak bands (the RNA concentration for
When comparing the bands of the Rat to the E. Coli (for other groups), there was a difference in bands. The Rat bands tended to be darker and have a brighter spot at the end of the gel. This means that there generally was a heavier Rat RNA concentration in the gels ran compared to the E. Coli. Also the banding pattern differed slightly. Possible reasons for this is the size of the ribosomes in eukaryotes and prokaryotes differ. Since the size differs, the RNA will be broken up into different banding patterns at different weights. Since the Rat RNA came from eukaryotic cells, which have bigger ribosomes, the banding pattern will tend to be brighter since there is more substance; hence the greater band intensity when compared to the E. Coli.