PCR protocols, troubleshooting and research tools
Troubleshooting
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1. PubMed: a service of the National Library of Medicine, includes over 15 million citations for biomedical articles back to the 1950's.

2. EMBO: The European Molecular Biology Organization

3. Nucleotides: The Entrez Nucleotides database is a collection of sequences from several sources, including GenBank, RefSeq, and PDB.

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Troubleshooting

 

 
 1. No PCR Product
 
- poor quality PCR primers or templates
- PCR reagent contamination
 
2. Primer dimer formation 
 
- if primer dimers form because of low level gene expression, increase the amount of template used
 
 3. Longer length nonspecific PCR products
 
- decrease the annealing time
- decrease the extension time and/or the extension temperature
- use less Taq polymerase
- design new primers
 
 4. Shorter nonspecific PCR products
 - increase the annealing temperature
- increase the annealing time
- increase the extension temperature
- decrease KCl buffer concentration
- use less Taq polymerase
- check the primer for repetitive sequences – redesign primer
5. Multiple bands on agarose gel (or multiple melting curve peaks) 
- note: agarose gel electrophoresis (or melting curve analysis) may not provide a completely accurate assessment of PCR specificity
- long amplicons have been known to generate more than one melting curve peak
- the presence of weak, shorter bands (than the expected band) may result from the migration of single stranded products – this does not necessarily indicate non-specificity
- when in doubt, use both agarose gel electrophoresis and melting curve analysis in combination to test the specificity of the reaction
6. PCR product abundance is low 
- verify that the PCR primers for mismatches
- increase the amount of PCR primer used
- use more DNA template
- use more Taq polymerase
- increase primer length by several nucleotides
7. The melting temperatures (Tm) of the primers are significantly different – what can be done? 
- the lower Tm can be raised by increasing the length of the primer
- add bases to the 3’ end

   

 

 

 

 

    
 

 

 

 
 
 
 
   
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