Methodology for sequencing double stranded DNA

Sequencing double stranded DNA using dye-labelled primer 1

Template DNA: The DNA is purified with NUCLEOBOND AX as described in the application guide. After precipitation with isopropanol (!) 2 ml of 70% ethanol are pipetted to the DNA pellet. The mixture is centrifuged 5 minutes at 8000 rpm. The wash solution is discarded and the pellet is dried thoroughly (speed vac, 50C, 1 minute). ATTENTION: Traces of ethanol lead to reduced peak heights, resulting in a decreasing reading rate. The glassy DNA pellet should be resuspended with 10 mM TE (pH 8) or ddH2O. For normal plasmid size (up to 10 kb) ~6 g plasmid DNA are necessary. Bigger plasmids (10-15 kb) give better sequencing results using a higher amount of plasmid DNA (~10 g).

Denaturing and annealing:
  10 l       template DNA (1/3 of a NUCLEOBOND AX miniprep, e.g. 5 ml XL-1 
              Blue/pBluescript/LB cultures)
  2 l        flourescently labelled primer (2-10 M)
  1 l        1M NaOH

Mix with a 10 l pipette, 65C, 3 min, place on 37C

Prepare A, C, G, T mixes used in one day needed for each reaction: 3 l respective termination mixes 1 l DMSO

Store on ice

Neutralization and termination: 1 l 1M HCl 2 l annealing buffer

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

1 l extension buffer 0.5 l T7 DNA polymerase

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

Stop: 4 l stop solution

Heat denature, load 4 l onto the sequencing gel

Note 1: In cooperation with the EMBL Institute in Heidelberg (Dr. Zimmermann, Prof. Ansorge) we developed a protocol for automated flourescent sequencing using NUCLEOBOND AX purified DNA.

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 with fl-15-dATP.

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

Back to the Nucleobond AX applications guide

Updated 1/20/98