DNA Polymerase Lab Report
OBJECTIVE
The purpose of this lab was to troubleshoot causes related to PCR components and develop an experiment that would test if the Taq amount is suitable for the PCR reaction to run correctly.
INTRODUCTION
Thermus aquaticus (Taq) DNA Polymerase is a bacterium that lives in thermophilic conditions and has been identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR (Noronha & Mullins, 1992). Why might you not be getting any bands on your PCR? If the Taq DNA polymerase concentration is too low, not all DNA fragments will be fully replicated, which in turn reveals very faint bands. The optimal enzyme concentration also depends on the amount of mystery meat (template DNA) used. …show more content…
The group hypothesized that Taq DNA Polymerase would add bases onto the end of an annealed primer during PCR (as shown in the molecular structure in Figure 2). This essentially causes Taq to interact with magnesium during the process, making it a cofactor of the polymerase.
Figure 2: Interpretation of Results
Seeing how one reagent is dependent on the other, PCR amplification will not occur if magnesium or Taq is left out of the reaction. Consequently, if we add excess Taq we will have to use an amount of MgCl2 that is above the concentration of Taq. This helps overcome inhibitors. DISCUSSION
I hypothesized that optimizing the Taq DNA polymerase concentration will enhance the overall performance of the PCR reaction. However, that proved to be wrong according to the experiment performed by the BABEC group. Their findings summarized:
I. Taq DNA Polymerase adds bases onto the end of an annealed primer, causing it to react with magnesium.
II. Magnesium is an important reagent.
III. Magnesium is a cofactor of the Taq polymerase
The purpose of this lab was to troubleshoot causes related to PCR components and develop an experiment that would test if the Taq amount is suitable for the PCR reaction to run correctly.
INTRODUCTION
Thermus aquaticus (Taq) DNA Polymerase is a bacterium that lives in thermophilic conditions and has been identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR (Noronha & Mullins, 1992). Why might you not be getting any bands on your PCR? If the Taq DNA polymerase concentration is too low, not all DNA fragments will be fully replicated, which in turn reveals very faint bands. The optimal enzyme concentration also depends on the amount of mystery meat (template DNA) used. …show more content…
The group hypothesized that Taq DNA Polymerase would add bases onto the end of an annealed primer during PCR (as shown in the molecular structure in Figure 2). This essentially causes Taq to interact with magnesium during the process, making it a cofactor of the polymerase.
Figure 2: Interpretation of Results
Seeing how one reagent is dependent on the other, PCR amplification will not occur if magnesium or Taq is left out of the reaction. Consequently, if we add excess Taq we will have to use an amount of MgCl2 that is above the concentration of Taq. This helps overcome inhibitors. DISCUSSION
I hypothesized that optimizing the Taq DNA polymerase concentration will enhance the overall performance of the PCR reaction. However, that proved to be wrong according to the experiment performed by the BABEC group. Their findings summarized:
I. Taq DNA Polymerase adds bases onto the end of an annealed primer, causing it to react with magnesium.
II. Magnesium is an important reagent.
III. Magnesium is a cofactor of the Taq polymerase