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3. Cycling termination reactions
Transfer 4.5µl of reaction mixture (prepared in step 2) to each termination
tube (‘G’, ‘A’, ‘T’ and ‘C’) from step 1. Mix well and overlay with 10-20µl of
mineral oil (if needed). Cap and place the tube in the thermal cycling
instrument.
Note: When sequencing single-stranded DNA, the primer may anneal to the
template with reduced specificity while the tubes are on ice, and extension of
these primers can occur as the thermal cycler heats up during the first cycle.
To minimize nonspecific extension products, the cycler can be pre-heated to
85-95°C or pre-cooled to 4°C.
4. Start the cycling program. Note: The specific cycling parameters used will
depend on the primer sequence and the amount and purity of the template
DNA. For the primers included in the kit and the suggested amount of purified
DNA (25-250fmol), cycle 30-60 times as follows:
dGTP dITP
95°C, 30s 95°C, 30s
55°C, 30s 50°C, 30s
72°C, 60-120s 60°C, 5-10min
(typically 30 cycles taking 2-3hr) (typically 30 cycles taking 3-5hr)
Fewer (1-10) cycles may produce better results when using 250-500fmol DNA.
5. Add 4µl of Stop Solution to each of the termination reactions, mix thoroughly
and centrifuge briefly to separate the oil from the aqueous phase.
Alternatively, remove 6µl from each termination reaction and transfer to a
fresh tube containing 3-4µl of Stop Solution. Samples should be kept on ice
for same day loading or may be stored frozen up to 3 days before loading onto
gel.
6. When the gel is ready for loading, heat the samples to 70°C for 2-10 minutes
and load immediately on the gel—3-5µl in each gel lane. Note: Heating in
open vials will promote evaporation of water from the formamide-reaction
mixture. This is not normally necessary, but will increase the signal by
concentrating the isotope and will promote more complete denaturation of the
DNA. This may improve results when using older
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P-ddNTPs. Avoid complete
evaporation to dryness by prolonged heating.