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9 thoughts on “P48 – Furia”
What in the figure shows that there was DNA degradation and what does this mean for the experiment’s results?
In “Figure 3: Quality Control Gel”, you can see the results of having run an agarose gel to determine if the DNA is degraded starting from the last three lanes for which Lane 1 is the DNA Ladder (10L), Lane 2 is phage DNA Sample 1 (25L), and Lane 3 is phage DNA Sample 2 (25L).
If you look, you can see a tight band of high molecular weight. This is smearing which indicates degraded DNA. As a result, this gel shows progressive degradation with increasing time of two newly isolated DNA samples run at 120V.
As for what this mean for the experiments’ results, it makes us unable to proceed to the restriction digest and PCR experiments. However, this may be resolved as degradation may be as a result of using very old DNA samples, using DNA extracted from formalin-fixed paraffin embedded samples, freezing and thawing DNA samples repeatedly, leaving DNA samples at room temperature, exposing DNA samples to heat or physical shearing, purifying DNA samples inefficiently so residual nuclease remain, etc. It is simply a matter of finding, and resolving the specific issue faced.
Were you unable to perform EM due to complications with the titer or a differing reason?
Correct, due to some yet unknown complications in calculating the titer of HTL, we were unable to preform electron microscopy.
However, we suspect there may have been an error in regards to the dilutions of the plaques, as the initial concentration of the plaques was too large at 10^-5 dilutions, as seen in “Figure 4: high titer lysate”. Therefore, in future experiments we suspect a dilution between 10^-5 and 10^-9 will yield significant results to be of use in electron microscopy.
Why was mycrobacterium smegmatis used as a host?
Mycobacterium smegmatis (M. smegmatis) was used as the host as it is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species.
Mycobacterium smegmatis (M. smegmatis) was used as a host as it is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species.
What does extra isolation do? Is it used to get more data for the sample?
The additional rounds of DNA isolation were preformed in order to reduce the number of plaques, as the original HTL was very concentrated, requiring the procedure to be repeated in order to achieve 20-200 plaques.