Other limitation of the experiment includes that there was no replication experiment that confirms the result of the experiment, the cross between the unknown mutants were limited, and that the growth of the colonies were left in the incubator for a long time so that determining growth was difficult. The first limitation that there is no replication. Replication of the experiment is important in determining what is happening is not due to chance or due to contamination. However, due to the limited amount of time, a replication was not performed. Another limitation is the cross between the unknown mutants were limited. While unknown alpha 1 and unknown alpha 2 was crossed with unknown a1 nd unknown a2, unknown alpha 1 and unknown a1 did not have the same genotype, as did unknown alpha2 and unknown a 2 because of this, unknown alpha 1 was not able to be crossed with unknown alpha 2 and unknown a2 is not able to be crossed with unknown a2. This limits the amount of analysis that can be done as it may be possible that both as have the mutation at the same gene but it was never shown because those two were never crossed. Another limitation is in determining the growth in MV and MV+adenine plates. Because the plates were left in the incubator for 3 days rather than 1 day like other plates, the growth was hard to determine because there were colonies that had noticeably less growth compared to others. Because those had less growth, it was hard to determine whether those counted as part of the colonies that grew well,especially the lack of growth was so noticeable compared to those that grew well.
Future experiments that could be done would be to replicate the experiment, fully cross all combination of mutants and genotype the yeast colonies. The first experiment that could be done is to replicate the experiment. By replicating the experiment, the certainty in the results can be increased, and the possibility that the results were skewed due to contamination or other factors would be limited. Another experiment that could be done is to cross the as and alphas with each other and themselves. By doing this, the genotype of the mutants can be better determined. For example, if unknown a 1 was crossed with unknown a1, and the resulting diploid colony grew well on YED and MV+adenine plate but poorly on MV plate, it can be determined that even if unknown a 1 does not have a mutation at ade1 or ade2, the mutation is still in the adenine pathway. Yet another experiment that can be done to confirm the site of mutation is to genotype the colonies. By extracting the DNA, running PCR and running the DNA on a gel, a more accurate result can be obtained. In addition, using the purified DNA, the DNA sequence can be determined, and the exact nature of the mutation, whether it is a spot mutation or a missense mutation can be determined.
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