Introduction Caenorhabditis elegans is a common organism that is used in many experiments due to its easy upkeep, low cost, short life cycle, fast reproduction, large reproduction size, and genetic manipulability with transparent body structure (Maxwell C. K. Leung, Phillip L. Williams, et al.). The use of C. elegans has allowed many discoveries to be made throughout history. One experimental technique that enhances their use is a green fluorescence protein (GFP), this allows different regions of the C. elegans to be highlighted for particular uses (Maxwell C. K. Leung, Phillip L. Williams, et al.). For one strain of C. elegans, SRU1, the GFP was fused to the promoter of CYP13A7 so it is considered a transcriptional fusion (Chakrapani, Baby
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elegans were placed on a 10 NGM-lite plate (3g/L NaCl, 17g/L agar, 2.5g/L peptone, 1mM CaCl2, 5μg/ml cholesterol, 1mM MgSO4, 25mM KPO4 buffer pH6.0) which contained 0P50 E. coli, one week prior so that they would reproduce. Two different stages of development were found on the plate, adult, and larva, and 2-5 worms of each stage was collected in 2μl of diluted water. The adults C. elegans and larva C. elegans were put in their own centrifuge tube with 10μl RNA buffer (RB) and then spun for 5 seconds. Both tubes were intubated after at 65°C for 10 minutes and then at 85°C for 1 minutes. They were both spun for another 5 seconds, following that 2μl DNase stop was added to each and they were incubated for 30 minutes at 37°C and spun for another 5 seconds. Each tube had 2.5μl RTmix added and were spun for 5 seconds before being placed in the thermocycler. In the thermocycler they were incubated at 25°C for 5 minutes, 42°C for 30 minutes, 85°C for 5 minutes, and then held at …show more content…
Elegans, strain SRU1, in the larva and adult stages is shown in table 1. The results were used to calculate the 2- Δ ΔCt which was used to determine which developmental stage had higher relative GFP gene expression. This was done by using the values in table1, first the ΔCTE was found by doing, 34.71-31.00 which is 3.71, and then ΔCTC was found from 33.94-30.72 which is 3.22. These values were used to find the ΔΔCt to be used in the final equation, that can be found by doing ΔCTE- ΔCTC which is .49. The result from 2- Δ ΔCt with the new value, .49 was plugged in, which is .71202509779. The findings proved that the larva, the experimental, has 29% less GFP gene expression than the adults, the control. These results show that there is different gene expression in different stages of development in C. elegans. This proves the hypothesis that the adult will have higher relative GFP gene expression compared to the larva stage. The gene expression in the larva and adults weren’t far off showing that they both have similar GFP gene expression in both stages of development. There were no embryos in the plate that was used for this experiment, so the comparison between adults and embryos couldn’t be concluded. The GFP in the C. elegans strain SRU1 is expressed in the cytoplasm since it is a transcriptional fusion, there might have been expression in the embryos as well but there would have still been more in the adults due to
elegans were placed on a 10 NGM-lite plate (3g/L NaCl, 17g/L agar, 2.5g/L peptone, 1mM CaCl2, 5μg/ml cholesterol, 1mM MgSO4, 25mM KPO4 buffer pH6.0) which contained 0P50 E. coli, one week prior so that they would reproduce. Two different stages of development were found on the plate, adult, and larva, and 2-5 worms of each stage was collected in 2μl of diluted water. The adults C. elegans and larva C. elegans were put in their own centrifuge tube with 10μl RNA buffer (RB) and then spun for 5 seconds. Both tubes were intubated after at 65°C for 10 minutes and then at 85°C for 1 minutes. They were both spun for another 5 seconds, following that 2μl DNase stop was added to each and they were incubated for 30 minutes at 37°C and spun for another 5 seconds. Each tube had 2.5μl RTmix added and were spun for 5 seconds before being placed in the thermocycler. In the thermocycler they were incubated at 25°C for 5 minutes, 42°C for 30 minutes, 85°C for 5 minutes, and then held at …show more content…
Elegans, strain SRU1, in the larva and adult stages is shown in table 1. The results were used to calculate the 2- Δ ΔCt which was used to determine which developmental stage had higher relative GFP gene expression. This was done by using the values in table1, first the ΔCTE was found by doing, 34.71-31.00 which is 3.71, and then ΔCTC was found from 33.94-30.72 which is 3.22. These values were used to find the ΔΔCt to be used in the final equation, that can be found by doing ΔCTE- ΔCTC which is .49. The result from 2- Δ ΔCt with the new value, .49 was plugged in, which is .71202509779. The findings proved that the larva, the experimental, has 29% less GFP gene expression than the adults, the control. These results show that there is different gene expression in different stages of development in C. elegans. This proves the hypothesis that the adult will have higher relative GFP gene expression compared to the larva stage. The gene expression in the larva and adults weren’t far off showing that they both have similar GFP gene expression in both stages of development. There were no embryos in the plate that was used for this experiment, so the comparison between adults and embryos couldn’t be concluded. The GFP in the C. elegans strain SRU1 is expressed in the cytoplasm since it is a transcriptional fusion, there might have been expression in the embryos as well but there would have still been more in the adults due to