Methodology for sequencing double stranded DNA

Sequencing double stranded DNA with fl-15-dATP

Denaturing and annealing:
  10 Ál     template DNA (1/3 of a NUCLEOBOND AX 20 prep, e.g. 5 ml XL-1 
            Blue/pBluescript/LB cultures)
  2 Ál      unlabelled primer (10-100 ÁM)
  1 Ál      1N NaOH

Mix with a 10 Ál pipette at 65C for 3 min, place on 37C

Prepare A, C, G, T mixes used in one day needed for each reaction:

3 Ál respective termination mixes 1 Ál DMSO

Store on ice

Neutralization and labelling: 1 Ál 1M HCl 2 Ál annealing buffer

Mix with a 10 Ál pipette (annealing for 15 min at 37C is optional, we don't think it is necessary, while others report improved signal strength)

1 Ál label mix 0.5 Ál T7 DNA polymerase

Incubate at 37C for 10 min.

Termination: 1 Ál extension buffer

Mix with a 10 Ál pipette, divide into 4 x 3.5 Ál, add to 4 Ál of the respective A, C, G and T mixes and incubate at 37C for 5 min.

Stop: 4 Ál stop solution; heat denature, load 4 Ál 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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Updated 1/20/98