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NE102 Final Lab Write-Up

5 ml tube to create the different restriction digests

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1.5 mL tube to create the different restriction digests. Agarose gel and Electrophoresis. After preparing the separate digests, we incubated them for an hour at 37º C. After the hour incubation period, we added 10 µL of Gel Loading Dye to each of the digests. In the electrophoresis we loaded five different wells with the DNA ladder, HpaI digest, HpaI/SmaI digest, BamHI digest, and BamHI/EcoRI digest respectively. 5 µL of the Gel Loading Dye was added to the first well. 20 µL of each of the digest reactions were added to wells two through four. We skipped a well and then added the same amounts in the same order. We set the SDS-PAGE to 80 volts and ran the digests for ninety minutes. We visualized our results over a UV box. Chemiluminescene. We added on 3 mL of the luminescent HRP substrate solution onto our blots after pouring out the TBST. We rocked the trays for one minute. Forceps were used to transfer the blots to the plastic folder, which was placed onto a film cassette. They were ready to be developed in the dark room using x-ray film. Fluorimaging.
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Following the last TBST wash for the immunoblots, we used a Fluorimager to detect the proteins in the ZnEgr1 and vector transfected cells. Figures Figure 1. Light micrographs of PC12 cells treated and untreated with NGF at objective of 20x and 40x. a) Undifferentiated PC12 cells at 20x. b) Undifferentiated PC12 cells before treatment with NGF at 40x. c) Differentiated PC12 cells after treatment with NGF at 20x. d) Differentiated PC12 cells at 40x. Figure 2. Light micrographs of the localization of Egr1 in PC12 cells with and without treatment of NGF. a) Egr1 in PC12 cells without treatment with NGF. b) The nuclei of the untreated PC12 cells dyed with DAPI. c) Egr1 in PC12 cells after one-hour treatment with NGF. d) The nuclei of the PC12 cells treated with NGF dyed with DAPI.
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Figure 3. Light micrographs of the localization of Neurofilament L (NFT L) in PC12 cells both treated and untreated with NGF. a) NFT L in PC12 cells without treatment of NGF. b) The DAPI- dyed nuclei of the PC12 cells without treatment of NGF. c) NFT L in PC12 cells after a two-day treatment of NGF. d) The DAPI-dyed nuclei of the PC12 cells with a two-day treatment of NGF. Figure 4. Image of the different regions of the protein after a restriction digest was added to the pGFP and pRas-G12V and the corresponding sizes in kilobases. It depicts the separate size fragments of the given plasmid and the gene of interest. Figure 5. a) Micrograph of PC12 undifferentiated cells transfected with pGFP at 20x. b) Micrographs of PC12 cells that underwent neuronal differentiation after being transfected with pGFP and pRas-G12V at 20x.
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Figure 6. Bar graph of the percentage of differentiated cells in cells transfected with just pGFP or pGFP plus pRas-G12V. Figure 7. Immunoblot of β-actin and Ras levels in GFP and GFP+Ras-G12V cell cultures after transfection with the expression plasmids.
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Figure 8. Light micrographs of differentiated and undifferentiated PC12 cells with a vector stable or ZnEgr1 stable. a ) Micrographs of undifferentiated PC12 cells stably- transfected with a vector and untreated with NGF. Taken at 20x. b) Micrographs of undifferentiated PC12 cells stably-transfected with a vector and untreated with NGF. Taken at 40x.
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5 mL tube to create the different restriction digests...

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