LICOR Terminator Sequencing Protocol

LICOR Terminator Sequencing Protocol
Written by Eda Malaj
Version 8-23-00
The LICOR dye-terminator sequencing reactions differ from the typical ABI BigDye-terminator sequencing reactions as instead of each dideoxynucleotide terminator containing a different fluorescent dye as is the case with the ABI Big-Dyes, the LICOR dideoxynucleotide terminators all contain the same dye label and thus each of the four terminator nucleotide reactions must be incubated and loaded separately. The advantage of these separate reactions and separate loadings is that with each of the four dideoxynucleotide terminator generated 'nested fragment sets' being physically separated, there is no need for the 'spectral deconvolution' that is required when all four reactions are combined, and thus longer read lengths are possible.

Note: The LICOR dye-terminators presently are in beta testing in our laboratory and are not commercially available as of yet either from LICOR or Amersham.

Prepare the master reaction mix in each of 8 wells (1-8 only) in row A and each of 8 wells (1-8 only) in row E of the Robbins 96 well cycle sequencing plate when there are 16 different sequencing templates. Note: if 8 or fewer sequencing templates, use only row A.
```
			Master Mix Component		Amount

			Template DNA			      1-2 ug
			Unlabeled Primer(6.5uM)		1.0 ul
			Reaction Buffer				1.5 ul
			Thermo Sequenase		    2.0 ul
			ddH20 to a final volume of	27.0ul
```

Thoroughly mix master mix well by pipetting up and down 3-4 times. Then spin down briefly and aliquot 6.5 ul of the master mix into each of the remaining wells as follows:

       three wells in rows B, C, and D, from the master mix in row A,
       three wells in rows F, G, and H, from the master mix in row E,
       for each of the original 16 samples, 1-8 in rows A and E, respectively

For each of the original 1-8, 16 samples from row A and E, respectively, add 2ul of the specific termination mix in each well, as follows:

       2ul of the Terminator Mix-A in the well A and E,
       2ul of the Terminator Mix-C in the well B and F,
       2ul of the Terminator Mix-G in the well C and G,
       2ul of the Terminator Mix-T in the well D and H,

Centrifuge the plate briefly.

Place the plate in the thermal cycler and start the LICOR sequencing program. This same LICOR cycle sequencing profile works for both primer and terminator reactions.

              95 deg C for 2 minutes
              95 deg C for 30 seconds
              50 deg C for 30 seconds
              72 deg C for 45 seconds
              72 deg C for 5 minutes
                4 deg C for soak

Remove the unicorporated dye-terminators by room temperature precipitation with 2-vol of ethanol as usual.
Dry the samples and add 3 ul of dye before loading.
Heat samples at 95 deg C for 3 minutes to remove any residual water, and load 1.0-1.5 ul in each well using the multichannel pipette.

Bruce Roe, broe@ou.edu