142S Primers

Table 1 contained the number of bands and the size of each band for each sample. The first lane, the molecular ladder contained five bands with molecular weights of 97kDa, 65kDa, 45kDa, 30kDa, and 14.4kDa, respectively from the top to the bottom the gel. The second lane, filtrate contained four bands with the molecular weight of 135kDa, 41.6kDa, 16.6kDa, and 11.2kDa, respectively from the top to the bottom. The third lane, S1 contained three bands with the

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

This was completed with over 65 tissue samples from various locations. The analysis was performed with quantitative PCR, using fluorescent signals.

The first agarose gel provided insight into whether or not Zassy’s genomic DNA had been cut by the Sal I HF (NEB) restriction enzyme. As seen in Figure 1, Zassy’s cut DNA, in lane 3 left clear fragments between the 3 kb and 0.5 kb areas. Zassy’s uncut DNA, in lane 2, left a clear and bright band above the 10 kb marker. Therefore, the gel confirmed that Zassy’s DNA was successfully cut with Sal I because there was a significant difference the cut and uncut DNA that was run through the gel. There was an additional lane of cut DNA because another student adopted Zassy in the early stages of this experiment. Nevertheless, the clear distinction between lanes 2 and 3 allowed the experiment to continue on to purification of the DNA.

60uL of each sample transferred to Eppendorf tubes, added with a 30uL sample buffer and heated at 95-degree Celsius for 4 minutes. The electrophoresis apparatus set up and filled with Tri-HEPES SDS running buffer. A 25uL of each sample loaded in this order from right to left: ladder, filtrate, S1, P1, P1 medium salt, P1 high salt, S2, P2, P2 medium salt, P2 high salt. The gel ran for 50 minutes at 110V and analyzed with UVP analyzer.

A PCR tube containing a Ready-To-Go™ PCR Bead was supplemented with 22.5 microliters of a solution containing TASR38-specific primers. 2.5µL of the mixture were added to the primer mixture, and the sample was stored in ice until the entire group had finished the process up to this point. The entire group’s samples of DNA were denatured for 20 seconds at 95°C, then incubated for 20 seconds at 64°C so that the primers could anneal, then incubated at 20 seconds at 72°C, and then polished for five minutes at

Table 4.14 and Table 4.15 shows the readings for the test conducted at pH8. It can be seen that there was very

The purpose of the DNA restriction and electrophoresis lab was to first become familiar with the properties of restriction enzymes and discover that they, along with agarose gel electrophoresis, are used to characterize DNA molecules. Restriction enzymes are used to make cuts or join together DNA fragments. The cuts result in either staggered cleavage, or blunt cleavage. Staggered cleavage results when the breaks are offset. This cleavage usually results in sticky end being produced because for each DNA end that is produced there are extensions with unpaired nucleotide bases. Blunt cleavage results when the breaks in the phosphodiester bonds are right across from each other.

Then it was centrifuged a second time which created P2 and S2. The results were shown in figure 1 and 2. Further analyzes showed that rubisco was likely present in S1 and in P2. Figure 1. P1 and S1 results Figure

The prepared samples were heated with 12.5% (v/v) β-mercaptoethanol for 5 minutes in a boiling water bath to denature the proteins. The SDS-polyacrylamide gel (Biorad, Any kD, Mini-PROTEAN TGX Gel, 10 well, 30 µl) was loaded with heated protein samples of crude extract, flow through, pre-dialysis, post dialysis, and molecular weight standard (Biorad). The gel was run at 24 mA until the tracking dye reached the bottom of the gel. The gel was removed and placed within a stain (0.4 mg/mL Coomassie Blue R250, 40% (v/v) methanol, 5% (v/v) acetic acid) until bands were

Figure 4: The 1/10 dilution is 10mL of Sewage water mixed in with 900mL of Turtle Creek water. The curves clearly show the closeness between positive control and DNA sample. Each sample has two repeats. Turquois is positive control, Red is the 1/10, and Green is negative

Agarose GE: Figure 1 shows the results of amplifying five types Arabidopsis thaliana fwa gene. successful PCR was a result of nice bands showing in figure 1. Wild type undigested (900bp), mutant undigested(860bp), and mutant digested (850bp) were all showing band in the same location. On the other hand, Wild type digested did not show any bands meaning amplification did not occur because McrBc digests fwa enzyme that is being methylated. Gel electrophoresis was used to determine the size of the base pairs from the logarithmic equation. Digested and undigested mutant genes showed hazy regions meaning they have less amount of amplification compared to wild type undigested which shows a significant amplification. No band was shown for

Good quality genomic DNA was isolated (Sambrook, et al 2002) and bothansA1 and ansA3 genes were amplified by PCR. Clear bands of both the genes showing a size of 1kb were observed under UV transilluminator after agarose gel electrophoresis (Fig 1).

Results of the first PCR reaction and gel electrophoresis with all primer sets can be seen in image 2. The first well contained the molecular weight marker, well 2; primer set 1, well 3; primer set 2, well 4; primer set 3, well 5; primer set 4 and well 6; primer set 5. The primer bands can be seen to have run to the end of the gel in wells 2 through 6, however no cDNA band were visible, only the weight maker bands were seen in the first well. The process was redone with a lower annealing temperature in the PCR stage with the aim to increase binding of the primers to the cDNA. Only primer set 2 was used, and again only marker bands in well 1 and primer bands at the end of well 2 were visible, which does not give any conclusive results, that can be seen in Image 2. Still determined, a third PCR reaction was performed with marker in well 1 and primer set 2 in wells 2 through 6, but with a PCR cycling conditions and temperature gradient. But, alas Image 3 revealed no useful data. As the GFP gene product was absent in al performed tests. Validation of extracted Zoanthid cDNA cannot proceed.