What is the sequencing protocol? The following protocol utilises ABI BigDye terminator v3.1 and ABI3730xl DNA analyser. 1. Sequencing Reaction Set-up: Sequencing reactions can be performed as follows: Please perform your sequencing reactions in 200 microliter centrifuge tubes. Component | Volume | Template DNA (300-600 ng) | x µL | Primer (3.2 uM) | 1 µL | ABI BigDye terminator v3.1* | 1 µL | 5x Sequencing Buffer | 3.5 µL | Milli-Q water | y µL | Total volume | 20 µL |
* The volume of terminator used is 1/8 reaction dilution. 2. Sequencing Cycling Conditions: Please use the following cycling conditions with a PE Applied Biosystems GeneAmp PCR System 9700 thermal cycler. If you have other thermal cycler, please consult Applied Biosystems (www.appliedbiosystems.com) for cycling conditions. 96 ° C 1 min 96 ° C 10s 50 ° C 5s 60 ° C 4 min 25 cycles 4 ° C (infinity)
3. Sample Submission After cycling reactions, seal the centrifuge tubes (containing 20 µL of reaction volume) with parafilm or equivalent tape. Mail your samples together with a copy of the order details to AgGenomics Pty. Ltd., Victorian AgriBiosciences Centre, 1 Park Drive, Bundoora, Victoria 3086, Australia. For sequencing protocol discussions, please call us on (03) 9479 3931 or email xpress@aggenomics.com.au |