Gradual or abrupt signal loss

 

Secondary structure - gradual - raw - website

Gradual signal loss

 

Secondary structure - gradual - electropherogram-GC-rich-website- 1

Gradual signal loss with continued base calling in a GC-rich region.

 

 

Gradual signal loss-GT repeats - electropherogram-website

Gradual signal loss with continued base calling after a GT-repeat region.

 

Secondary structure-abrupt-raw-website

Abrupt signal loss

 

Secondary structure-abrupt-electropherogram-website

Abrupt signal loss with no base calling.

 

 

Poor base calling-GT-repeats - electropherogram-website

Abrupt signal loss with poor base calling after a GT-repeat region.

 

Possible causes

Recommendations

Secondary structures (GC and AT rich templates can cause DNA to form hairpins)

Sequence the complementary strand.

Re-sequence with 5% DMSO.

Repetitive regions (GC and GT repeats can cause a decrease in signal due to depletion, slippage or secondary structure)

Try a primer that anneals in a different position.

Re-sequence with 5% DMSO.

siRNA template

Re-sequence with 5% DMSO.

If signal loss early in the sequence (within the first 100 nucleotides): primer-dimer formation

Ensure the primer does not anneal to itself.

Ensure there are no contaminating PCR primers (i.e. cleaned up PCR product)