Denaturing and annealing: 10 µl template DNA (1/3 of a NUCLEOBOND AX miniprep, e.g. 5 ml XL-1 Blue/pBluescript/LB cultures) 2 µl flourescently labelled primer (2-10 µM) 1 µl 1M NaOHNote 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.
Mix with a 10 µl pipette, 65C, 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 termination: 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 extension buffer 0.5 µl T7 DNA polymerase
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 with fl-15-dATP.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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