User:Saroj Pandey/Notebook/SNP PCR optimization/2014/10/06

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gradient PCR for optimization

• Correct primer combinations from previous result were again used for a gradient PCR to find out the optimal annealing temperature

gradient PCR
gradient PCR
electrophoresis
electrophoresis
electrophoresis
electrophoresis

Primers

1. Product size: 510bp [gaF + Gfb(R)]

gaF: ATCCGTGATGCTGTGCTATG

Gfb(R): CAATCACTGTTGCTCAGTGC

2. Product size: 510bp [gaF + Cfb(R)]

gaF: ATCCGTGATGCTGTGCTATG

Gfb(R): CAATCACTGTTGCTCAGTGG

3. Product size: 235bp [Cfb(F) + Gfb(R)]

Cfb(F): GGTGGCAACCAGGTCTTTAG

Gfb(R): CAATCACTGTTGCTCAGTGC

4. Product size: 235bp [Cfb(F) + Cfb(R)]

Cfb(F): GGTGGCAACCAGGTCTTTAG

Cfb(R): CAATCACTGTTGCTCAGTGG


Observation

• Taster [gaF + Cfb(R)]: Expected products were seen at different temperatures except 58.1°C and 56.4°C where bands were seen at 100bp. A faint band was seen at 68°C which was not observed at 69°C.

• Non-taster [gaF + Gfb(R)]: Expected product size of 510bp was seen at temperatures 66.3°C and below and not observed above it.

• Taster [Cfb(F) + Cfb(R)] and Non-taster [Cfb(F) + Gfb(R)]:: Expected product size of 235bp was seen from 55.5°C to 63.7°C and became fainter above it. All the reactions, including blanks, have side products below 100bp which might be primer dimers.



Conclusion

• The optimal annealing temperature for all four reactions seems to be 60.5°C.



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