User:Saroj Pandey/Notebook/SNP PCR optimization/2014/09/25: Difference between revisions

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* A PCR using 4 primers (@ outer and 2 inner primers) was performed
* A PCR using 4 primers (@ outer and 2 inner primers) was performed
* Forward inner primer ends in G and reverse inner primer ends in C
* Forward inner primer ends in G and reverse inner primer ends in C
* Expected band sizes for taster: 992bp and 510bp
* Expected band sizes for taster: 992bp and 522bp


[[Image:multiplexPCR.JPG]]
[[Image:multiplexPCR.JPG]]
[[Image:multiplexPCR_26.09.jpg|right|thumb|350px|Electrophoresis]]
[[Image:multiplexPCR_26.09.jpg|right|thumb|350px|Electrophoresis]]
Observation:
• No bands are seen at 992bp which tells that amplification with the outer primers was not successful
• Faint bands are seen at about 500bp in both the taster and the non-taster
• As observed in previous PCRs, a thick bright band is seen below 100bp. But there is also another band at approximately 150bp.
Conclusion:
• Since the expected products are of similar lengths, this PCR is unable to distinguish between the two variants and other set of primers could be used




Revision as of 04:01, 1 October 2014

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Allele Specific PCR and Multiplex PCR

  • A PCR using 4 primers (@ outer and 2 inner primers) was performed
  • Forward inner primer ends in G and reverse inner primer ends in C
  • Expected band sizes for taster: 992bp and 510bp
  • Expected band sizes for taster: 992bp and 522bp

Electrophoresis


Observation:

• No bands are seen at 992bp which tells that amplification with the outer primers was not successful

• Faint bands are seen at about 500bp in both the taster and the non-taster

• As observed in previous PCRs, a thick bright band is seen below 100bp. But there is also another band at approximately 150bp.


Conclusion:

• Since the expected products are of similar lengths, this PCR is unable to distinguish between the two variants and other set of primers could be used




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