3.Cycling termination reactionsTransfer 4.5∝l of reaction mixture (prepared in step 2) to each termination tube (‘G’, ‘A’, ‘T’ and ‘C’) from step 1. Mix well and overlay with 10-20∝l 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 4∝l of Stop Solution to each of the termination reactions, mix thoroughly
and centrifuge briefly to separate the oil from the aqueous phase. Alternatively, remove 6∝l from each termination reaction and transfer to a fresh tube containing 3-4∝l 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—3-5∝l 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 33P-ddNTPs. Avoid complete evaporation to dryness by prolonged heating.