To set up genotyping reactions, we make up a mixture of Hi Di (formamide) and Rox size standard. Currently, adding 20 ul of the Rox size standard to 1 ml of Hi-Di works well. This mixture can then be aliquoted 9 ul / well into a skirted plate that fits the ABI sequencer. We are currently using TempPlateIII from USA Scientific p/n 1402-9700. Finally, add 1 ul of your genotyping PCR reaction to each well. Seal with septa seal, mix and spin down. It's ready to run.
ABI has undergone some changes in filter sets as they introduce new sequencers. Here is some information on the dye sets for various machines and their peak emission wavelengths.| Machine | 377 | 3100 | 3730 |
| Dye set | D | DS-33 | |
| 1 | 6FAM (518 nm) | 6FAM (518 nm) | 6FAM (518 nm) |
| 2 | TET (538 nm) | HEX (556 nm) | VIC (554 nm) |
| 3 | HEX (556 nm) | NED (575 nm) | NED (575 nm) |
| Size | TAMRA (576 nm) | ROX (605 nm) | PET (595 nm) |
Previously on the 377, we used Tet dye (yellow) to label primers for genotyping. However, there is no filter for this dye on the new machines and it falls between the 6FAM and Hex filter and will bleed into Hex. While it can be used by itself (and will come out in the hex (green) channel) it can not be multiplexed with Hex labeled primers. Since Hex and Vic are interchangeable, we can keep using our Hex labeled primers. However, Ned is a proprietary ABI dye so it is not obvious what to use for this channel.
Below are the emission spectra for dyes used on the 3100 (filter set D) and the 3730 (filter DS-33). This graph comes from ABI