The tables provided below encompass the majority of issues and corresponding solutions encountered during PCR experiments.

To save time on PCR setup optimization, it is recommended to use Ammonium Buffer for most PCR applications. Ammonium Buffer is a highly reliable 10x PCR buffer that yields a high quantity of PCR products while minimizing the need for optimization of Mg2+ concentration and/or annealing temperatures.

 

PCR product does not have the correct size

POSSIBLE CAUSES
 SOLUTIONS
Contamination by nucleases Try again with fresh reagents
Mis-priming Test that primers do not have additional complementary regions within the template DNA
Non optimal MgCl2 concentration Adjust MgCl2 concentration as advised in product datasheet
Non optimal annealing temperature Retest Tm values of primers

 

Absence of PCR product

POSSIBLE CAUSES SOLUTIONS
Low primer specificity Verify that primers are complementary to the correct target sequence
Too low primer concentration Adjust in the range 0.1 – 1 µM
Suboptimal reaction conditions Optimise annealing by running a temperature gradient
Adjust MgCl2 concentration as advised in product data sheet
Poor template quality Test DNA using gel electrophoresis before and after addition of MgCl2
Check 260/280 ratio of DNA template
Missing a reaction component Make a new PCR mix
Inhibitors in the reaction Ensure that template DNA is purified or decrease sample volume
PCR run is not optimal Add more cycles
Recheck the PCR program
Recalibrate heating block
Your template or target is complex For GC-rich sequences or other complex DNA targets optimize conditions using GC-rich Target kit

 

Smears or multiple bands on the gel

POSSIBLE CAUSES SOLUTIONS
Premature replication Use TEMPase Hot Start DNA Polymerase instead
Set PCR reaction up on ice
Too low annealing temperature Increase annealing temperature
If not already using Ammonium Buffer, then shift to this buffer
Excess primers Adjust in the range 0.1 – 1 µM
Non optimal MgCl2 concentration Adjust MgCl2 concentration as advised in product datasheet
Non optimal primer design Ensure that primers are non-complementary
Increase length of primers
Avoid GC-rich 3’ ends
Contamination with non-template DNA Always use filer tips, PCR grade water
Use separate areas for PCR reaction setup, DNA preparation, PCR thermal cycling and gel electrophoresis
Incorrect template concentration Adjust template concentration as advised in product datasheet

 

Sequence errors

POSSIBLE CAUSES SOLUTIONS
Low fidelity polymerase Use AccuPOL DNA Polymerase with Ammonium Buffer
Template DNA has been damaged Prepare a new DNA template
Limit the exposure of template DNA to UV
Lower initial heating time
Suboptimal reaction conditions Decrease extension time
Decrease MgCl2 concentration
Lower the amount of cycles
Problems with nucleotide composition Make a fresh solution of nucleotide mix

 

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