Denaturing and annealing:
10 µl template DNA (1/3 of a NUCLEOBOND AX 20 prep, e.g. 5 ml XL-1
Blue/pBluescript/LB cultures)
2 µl unlabelled primer (10-100 µM)
1 µl 1N NaOH
Mix with a 10 µl pipette at 65C for 3 min, place on 37C
Prepare A, C, G, T mixes used in one day needed for each reaction:
3 µl respective termination mixes
1 µl DMSO
Store on ice
Neutralization and labelling:
1 µl 1M HCl
2 µl annealing buffer
Mix with a 10 µl pipette (annealing for 15 min at 37C is optional, we don't
think it is necessary, while others report improved signal strength)
1 µl label mix
0.5 µl T7 DNA polymerase
Incubate at 37C for 10 min.
Termination:
1 µl extension buffer
Mix with a 10 µl pipette, divide into 4 x 3.5 µl, add to 4 µl of the
respective A, C, G and T mixes and incubate at 37C for 5 min.
Stop:
4 µl stop solution; heat denature, load 4 µl onto the sequencing gel
For solutions and buffers for sequencing with T7 DNA Polymerase or for more information please refer to Sequencing Solution Buffer Preparation and Sequencing Double Stranded DNA using dye-labelled primer.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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