User:Saroj Pandey/Notebook/SNP PCR optimization/2014/09/20

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Gradient PCR (SNP)

• As the selected primers were not able to differentiate two different DNA templates, another approach was carried out to conduct the PCR in a range of different annealing temperatures

• Gradient PCR was done with annealing temperature ranging from 56 °C to 70 °C

Image:gradient_PCR.JPG

Electrophoresis
Electrophoresis
Electrophoresis
Electrophoresis


Observations:

• With G (ff) primers, products were seen at expected band length (161 bp) at all temperatures with the most prominent at 57 °C and faint bands above 68 °C. Unspecific products just below 400 bp were seen in 4 samples at lower temperatures.

• With G (fb) primers, expected product size (235 bp) was observed in 6 out of 8 samples. Brightest band was seen at 57 °C which became fainter with increasing temperature and no bands were seen above 68 °C. No unspecific products were seen.

• Unspecific products were much less (only one or none) as compared to previous experiment


Conclusion:

• For the set of primers used here, 57 °C seems to be the optimal annealing temperature

• The primers are unable to differentiate between two templates



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