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After the cleanup and the completion of the

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After the cleanup and the completion of the electrophoresis, the gel was taken out of the buffersolution and a UV transilluminator revealed the migrated bands. After photographing the gelsunder UV light, the gel buffer was properly discarded, and the experiment was complete.ResultsIn this experiment, the PCR product was determined and then verified if the correct onewas created following amplification. When compared to a standard curve from gelelectrophoresis, the existence of the true PCR product was verified.Using the Snapgene program, it is possible to find an estimate base pair size for the GFP geneand the added primers. The figure below showcases how with the primers and the amplificationprocess, the DNA product will be about 757 base pairs in size.
Dholaria 8Figure 1: Predicted PCR Product Base Pair SizeFigure 1. The figure above showcases the estimate PCR product base pair size which can be used to verify the gel electrophoresis. From theSnapgene program, it is estimated that the product will be about 757 base pairs assuming complete annealing of the primers.To test if the PCR Product will ligate with the pRSET plasmid, the Snapgene program wasutilized. Using the restriction enzymes PstI for the forward primer and HindIII for the reverseprimer, recombination of the PCR product was determined to be possible.Figure 2: Ligation of amplified GFP product with pRSET plasmidFigure 2. The figure above shows the ligation ofthe forward primer to the pRSET plasmid byutilizing the PstI restriction enzyme. Though thereverse primer region is not visualized in thefigure, HindIII was utilized to fixate the PCRproduct onto the vector.
Dholaria 9After electrophoresing the PCR product for 1 hour at about 100 V,a band fluoresced between 500 base pairs and 1000 base pairs. Theexpected size of the product was found to be 757 base pairs fromSnapgene. Seeing that the band appeared in between the two ladderbands and the fluorescing band was found closer to the 500 base pairsize marker, it is reasonable to assume that the desired PCR productwas obtained.Figure 4: Graphical Representation of DNA Size Markerslog (࠵?࠵?࠵?) = −0.0014(1365 ࠵?࠵?࠵?࠵?࠵?࠵?) + 1.7527 = −0.1583࠵?࠵?࠵? = 10<=.>[email protected]= 0.695࠵?࠵? = 0.695 ∗ 1000 = 695࠵?࠵?࠵?࠵?࠵?࠵?࠵? ࠵?࠵?࠵?࠵?࠵? =|LMNOPQOR<OSTOUVOR|OSTOUVOR∗ 100 =|WX?<Y?Y|Y?Y∗ 100 =8.19%After receiving the electrophoresis results, the migration distance of the PCR product band wasdetermined in pixels (photograph). The estimated migration distance was found to be 1365Figure 3: GelElectrophoresisOf PCRProductFigure 4. The graph above represents the logarithm of kilobase pairs vs. themigration (number of pixels) that each band corresponds to from figure 3.Figure 3. The figure aboveshowcases the result of the gelelectrophoresis. Lane 1 shows theDNA marker sizes and Lane 2shows the PCR Product.

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