204
KLAUS DEICHMANN
- Loss of activity of the polymerase? Always store the enzyme at -20°C,
dilute just as much as you need and keep the dilution on ice until use.
Use the diluted enzyme as quickly as possible. Beware of so-called
frost-free freezers which warm up above 0°C daily!
- Labeled nucleotides may be too old. 32 P should be less than 2 weeks
old, 35 S Iabel can be kept at -20°C for up to 6 weeks.
• Diffuse or fuzzy bands throughout the lanes
- Bad template DNA, contaminated with protein?
- Polyacrylamide gel of poor quality? Always use electrophoresis grade
reagents and prepare fresh solutions and buffers in all critical experiments.
- Gel run too hot/too cold? Adapt the voltage. Gels should be run at a
temperature of about 50-55°C.
- Sampies incompletely denatured? Always heat the samples to at least
75°C and place on ice prior to loading the gel.
• Bands through all 4 lanes at the same position; above this site faint
bands
- This is most probably due to strong secondary structures of the template. The enzyme will pause at these structures. The underlying sequences most probably contain dG and dC rich residues and/or dyad
symmetries where hairpin loops are formed. This means that all extensions in the termination reaction will stop at the same position, and
the enzyme will fall off the forming strand, no matter which of the 4
ddNTPs has been used. Try to sequence the template on both strands,
lower the dNTP concentration in the termination reaction, reduce the
reaction time to 1-2 min and raise the amount of template DNA to
keep the enzyme from reaching the structure. Y ou can add E. coli single strand binding protein (USB) to the reaction. This may prevent the
DNA from forming secondary structure. However, the protein has to
be removed by proteinase K prior to loading the samples on the gel. lt
is best to use Taq polymerase sequencing, as high temperature during
the extensionwill prevent the template from forming secondary structures.
• Bands at the same position in 2 or 3 lanes at specific regions
- This is most probably due to compression artifacts. These occur, when
secondary DNA structures of the polymerized strands, especially
those containing dG and dC rich residues, do not remain fully denatured under the conditions of electrophoresis. Try to substitute dGTP
with diTP or 7-deaza-dGTP during the sequencing reactions. How-
KLAUS DEICHMANN
- Loss of activity of the polymerase? Always store the enzyme at -20°C,
dilute just as much as you need and keep the dilution on ice until use.
Use the diluted enzyme as quickly as possible. Beware of so-called
frost-free freezers which warm up above 0°C daily!
- Labeled nucleotides may be too old. 32 P should be less than 2 weeks
old, 35 S Iabel can be kept at -20°C for up to 6 weeks.
• Diffuse or fuzzy bands throughout the lanes
- Bad template DNA, contaminated with protein?
- Polyacrylamide gel of poor quality? Always use electrophoresis grade
reagents and prepare fresh solutions and buffers in all critical experiments.
- Gel run too hot/too cold? Adapt the voltage. Gels should be run at a
temperature of about 50-55°C.
- Sampies incompletely denatured? Always heat the samples to at least
75°C and place on ice prior to loading the gel.
• Bands through all 4 lanes at the same position; above this site faint
bands
- This is most probably due to strong secondary structures of the template. The enzyme will pause at these structures. The underlying sequences most probably contain dG and dC rich residues and/or dyad
symmetries where hairpin loops are formed. This means that all extensions in the termination reaction will stop at the same position, and
the enzyme will fall off the forming strand, no matter which of the 4
ddNTPs has been used. Try to sequence the template on both strands,
lower the dNTP concentration in the termination reaction, reduce the
reaction time to 1-2 min and raise the amount of template DNA to
keep the enzyme from reaching the structure. Y ou can add E. coli single strand binding protein (USB) to the reaction. This may prevent the
DNA from forming secondary structure. However, the protein has to
be removed by proteinase K prior to loading the samples on the gel. lt
is best to use Taq polymerase sequencing, as high temperature during
the extensionwill prevent the template from forming secondary structures.
• Bands at the same position in 2 or 3 lanes at specific regions
- This is most probably due to compression artifacts. These occur, when
secondary DNA structures of the polymerized strands, especially
those containing dG and dC rich residues, do not remain fully denatured under the conditions of electrophoresis. Try to substitute dGTP
with diTP or 7-deaza-dGTP during the sequencing reactions. How-