Attention: Please contact Katie Carter if your template is >20kb or very G-rich, as the following protocol may need to be altered.
If you are submitting >48 samples at a time, please use a PCR plate that is compatible with our ABI 3730 sequencer. The following will work: Cat. # T-3085-1 from ISC BioExpress or Cat. # N801-0560 from Life Technologies. In each well of a 96-well PCR plate, put 5 uL of template/primer mixture, using half the amounts of template and primer listed for standard sequencing. (The amount requested for standard sequencing is actually double what is needed.)
Next make a master mix based on the following volumes of reagents per well: 2 uL deionized water
1 uL Big Dye Terminator 3.1 (thaw on ice)
1.5 uL 5X buffer
0.5 uL DMSO
Now add 5 uL of master mix to each well, making sure not to cross contaminate.
Place a PCR sealing mat or film on the plate and vortex plate slightly at low speed. Centrifuge for a few seconds to get the mixture down to the bottom of each well. We recommend sealing film (Cat. #: 48461) from Edge BioSystems to prevent sample evaporation. Many other brands do not seal very well or are too sticky to remove.
Using a thermal cycler, heat the plate at 96°C for 5 minutes. Next, subject the plate to 25 cycles under the following conditions:
96°C for 30 seconds
50°C for 15 seconds
60°C for 4 minutes
Bring plate to the Core Labs and place in the refrigerator (not the freezer!). Make sure sample names are clearly marked on the plate. Submit web-order, indicating "Economy" or "Economy Plate" under sequence type. When filling in the web-order, please submit the samples in the following order: A1, B1, C1 ... H1, A2, B2, etc.
Notify Katie Carter at least a day in advance before bringing plates to the Core Labs, as samples should be cleaned within 24 hrs of running extension reactions.