Optimizing the magnesium chloride concentration in a polymerase chain reaction / Sazaliza Mat Isa
The polymerase chain reaction (PCR) is any enzymatic method of synthesizing or amplification of large quantities of a targeted region of DNA in vitro. When developing a protocol for PCR amplification of a new DNA target, it may be important to optimize the reagent concentrations, cycling temperature...
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| Format: | Student Project |
| Language: | English |
| Published: |
2007
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| Subjects: | |
| Online Access: | http://ir.uitm.edu.my/id/eprint/47508/1/47508.pdf http://ir.uitm.edu.my/id/eprint/47508/ |
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| Summary: | The polymerase chain reaction (PCR) is any enzymatic method of synthesizing or amplification of large quantities of a targeted region of DNA in vitro. When developing a protocol for PCR amplification of a new DNA target, it may be important to optimize the reagent concentrations, cycling temperatures and cycle numbers. Magnesium chloride (MgCb) had been shown to influence the primer annealing temperature, fidelity, specificity and yield of a PCR run. In this study, variable concentration of MgCla have been used to determine its effects on a PCR performance and yield. The template DNA, reaction buffer, dNTPs, primers and DNA polymerase were not changed throughout the study. The PCR amplicons were then electrophoresed, stained and then photographed using the UV light. Results showed that at 0.5 mM MgCb concentration, no DNA band was produced in the gel. However, with an increased or excess in MgCh concentration (3.0 mM to 6.0 mM) multiple bands of non-specific products were formed and band intensity decreased. The optimal MgCb concentration was found to be 1.5 mM. At this optimized PCR amplification, the DNA band appeared as a single, bright band. For this and other reasons, it is deem necessary to optimize specific PCR amplification with respect to this divalent cation. |
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