What are the cycle sequencing parameters I should use for the GeneAmp PCR System?
To set up cycle sequencing reaction with the GeneAmp PCR System (2400, 9600, 9700) with BigDye 3.1, use the follow parameters (Adapted from PE Biosystems): Step Sequencing reaction-Plasmid/PCR products Sequencing reaction BAC/Cosmic Clones 1 10 ul Reaction Volume. 20 ul Reaction Volume. 2 Heat the tubes at 96 oC for 1 minute Heat the tubes at 96 oC for 5 minutes Repeat the following for 30 cycles Repeat the following for 50 cycles *Rapid thermal ramp to 96 oC *Rapid thermal ramp to 96oC *96 oC for 10 sec *96 oC for 30 sec *Rapid thermal ramp to 50 oC *Rapid thermal ramp to 50 to 55 oC *50 oC for 5 sec *50 to 55 oC for 10 sec *Rapid thermal ramp to 60 oC *Rapid thermal ramp to 60 oC *60 oC for 4 min *60 oC for 4 min 3 Rapid thermal ramp to 4oC and hold until ready to purify. 4 Spin down the contents of the tubes in a microcentrifuge. 5 Proceed purification step (Laragen provides free BigDye Purification) Note: Rapid thermal ramp is 1oC per sec.
