GENOTYPING ON THE 3730
Dye Information:
Dyes supported on the 3730: 6-FAM, VIC, NED, PET
Ladder: LIZ 500 bp size standard
6-FAM = Blue
VIC = Green
NED = Yellow (Black)
PET = Red
LIZ = Orange
Dye ordering info:
6-FAM is a non-proprietary dye available from most primer synthesis companies (IDT,
Invitrogen, etc.)
VIC, NED & PET are ABI proprietary dyes and must be ordered through the company.
HEX can be used in place of VIC (they are the same dye color). HEX is a non-proprietary
dye available through IDT, Invitrogen, etc. Caveat: The sensitivity of the machine
to HEX is poor, thus peak heights theoretically will be greatly reduced compared to
FAM, NED and PET. The DNA concentration of HEX amplified products can be
adjusted to compensate (i.e. load more HEX-labeled product than other
amplification products; this needs to be optimized for your system).
DNA concentration and multiplexing:
The 3730 is set-up to support multiplexing of different dye colors (i.e. combine
amplification products labeled with different dyes in the same well). Amplification
products of the same dye color with very different product lengths (requires a priori
knowledge of product length) can be multiplexed as well.
Additionally the concentration of amplified product is usually much greater than the
sensitivity of the machine (will result in “blow-out” of peak heights), requiring a dilution
of PCR product.
Thus, amplified product can be combined and multiplexed in one step (I refer to this as
the dilution plate), with a second dilution to combine with ladder (this is the 3730 plate).
The optimal dilution needs to be experimentally determined, but a good place to start is
with a 1:200 dilution (1:20 in the first step, 1:10 in the second).
Combine 1ul of each PCR product (i.e. 4ul total product for 4 PCR products) with water
to a total volume of 20. In the second dilution combine per well, 1 ul combined, diluted
product with 0.1ul ladder to a total volume of 10. Always load 10ul total volume onto the
3730 plate.