Cycle sequencing reaction Sample Clauses

Cycle sequencing reaction. Initially primers and Big Dye Terminator mix were defrosted. The cycle sequencing reaction was prepared by mixing 1.5 μl of BigDye Terminator sequencing Buffer, 1 μl of BigDye Terminator mix v3.1 and 4.5 μl of distilled water. The sequencing reaction was aliquoted and 7 μl of sequencing reaction mix were pipetted into the allocated sequencing plate xxxxx. 1.2 μl of forward sequencing primer were added to the allocated column and the same process was followed for the reverse primer. Using the plate spinner, the PCR plate was span to draw condensation to the bottom of the xxxxx; then the plate was placed on a magnetic rack and the sealer was removed. Using a multichannel pipette 2 μl of PCR product were added to each well. The plate was covered and span. The sequencing mixture was temperature cycled as per protocol listed in Table 5. Temperature (oC) Time (minutes) Number of cycles 96 1 55 0.25 35 60 1 60 5 1 Table 5: Cycle sequencing parameters