LICOR Terminator Sequencing Protocol
Written by Eda Malaj
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
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, firstname.lastname@example.org