Restriction Mapping of Plasmid DNA
Preparing 5 Eppendorf Tubes
All tubes contain 1 μL of the plasmid DNA, and 2 μL of 10X buffer #2. Tube 1 is the control so there is no enzyme added only 17 μL DI water. For tube 2 the DNA is mixed with 0.5 μL of BamHI and 16.5 μL of DI water, to see total number of base pairs from the linear DNA. In tube 3 DNA is digested with 2 restriction enzymes, which can give the relative distance between the two enzymes. To be more specific, 0.5 μL of Bam HI and EcoRI were added with the reminder of 16 μL of DI water added as well. In tube 4 and 5 it is the same concept as tube 3, the only difference is that tube 4 has BamHI and HinDIII restriction enzymes and Tube 5 has HinDIII and EcoRI restriction enzymes. After …show more content…
The 50 mL of 0.8% agarose gel is heated in the microwave for 1.5 minutes. The solution was cooled to room temperature and 5 μL of GelStarTM, a DNA marker, was added to the 50 mL. The DNA fragments will bind to GelStarTM and the GelStarTM enables the DNA complex to be visible under UV light. Carefully pour the solution into the gel-casting tray, insert the well comb and let the gel solidify. Place the gel tray so that it is parallel with the gel box and then fill the chamber of the gel box with 1XTBE until the buffer completely covers the …show more content…
The figure 2 shows the Agarose Gel Electrophoresis sample run that contained the GeneRuler and tubes 1-5. The first well contained the GeneRuler and the second well contained PAMP which was filled with Plasmid DNA without the restriction enzymes. The third well contained DNA with one restriction enzyme BamHI that gave one fragment of DNA. The fourth well contained DNA with two restriction enzymes, BamHI and EcoRI, that gave off two fragments. The fifth well contained DNA with two restriction enzymes, BamHI and HinDIII, that gave off three fragments but the second fragment was too weak to be counted as a fragment. The sixth well contained DNA with two restriction enzymes, HinDIII and EcoRI, that gave off two fragments as seen by the figure
Preparing 5 Eppendorf Tubes
All tubes contain 1 μL of the plasmid DNA, and 2 μL of 10X buffer #2. Tube 1 is the control so there is no enzyme added only 17 μL DI water. For tube 2 the DNA is mixed with 0.5 μL of BamHI and 16.5 μL of DI water, to see total number of base pairs from the linear DNA. In tube 3 DNA is digested with 2 restriction enzymes, which can give the relative distance between the two enzymes. To be more specific, 0.5 μL of Bam HI and EcoRI were added with the reminder of 16 μL of DI water added as well. In tube 4 and 5 it is the same concept as tube 3, the only difference is that tube 4 has BamHI and HinDIII restriction enzymes and Tube 5 has HinDIII and EcoRI restriction enzymes. After …show more content…
The 50 mL of 0.8% agarose gel is heated in the microwave for 1.5 minutes. The solution was cooled to room temperature and 5 μL of GelStarTM, a DNA marker, was added to the 50 mL. The DNA fragments will bind to GelStarTM and the GelStarTM enables the DNA complex to be visible under UV light. Carefully pour the solution into the gel-casting tray, insert the well comb and let the gel solidify. Place the gel tray so that it is parallel with the gel box and then fill the chamber of the gel box with 1XTBE until the buffer completely covers the …show more content…
The figure 2 shows the Agarose Gel Electrophoresis sample run that contained the GeneRuler and tubes 1-5. The first well contained the GeneRuler and the second well contained PAMP which was filled with Plasmid DNA without the restriction enzymes. The third well contained DNA with one restriction enzyme BamHI that gave one fragment of DNA. The fourth well contained DNA with two restriction enzymes, BamHI and EcoRI, that gave off two fragments. The fifth well contained DNA with two restriction enzymes, BamHI and HinDIII, that gave off three fragments but the second fragment was too weak to be counted as a fragment. The sixth well contained DNA with two restriction enzymes, HinDIII and EcoRI, that gave off two fragments as seen by the figure