Resources » Technical Resource Centers » Gene Technical Resources » Molecular Cloning Center » PCR Troubleshooting Guide
In molecular cloning, after the synthesis of cDNA from mRNA molecule templates, a PCR program must be designed to amplify the gene of interest, as well as add additional elements such as restriction sites or detection/purification tags. Intrinsic properties of gene sequences such as high GC content, long stretches of the same polynucleotide, and sequences encoding hairpin loop structures can all hinder PCR efficiency.
Use the troubleshooting guide below to identify the cause of PCR failure and improve PCR efficiency or get your desired gene in the vector you want the easy way with GenEZ™ ORF clones. Start with a search for your gene.
Problem | Causes | Solutions |
---|---|---|
Low or no PCR product yield
|
Number of PCR cycles is insufficient
|
|
Template is degraded
|
|
|
Template is contaminated with PC inhibitors
|
|
|
Thermocycler program annealing and extension temperatures are not optimal
|
|
|
Reaction is missing Taq polymerase or other reaction component
|
|
|
Primer concentration too low
|
|
|
Target sequence is not in DNA template
|
|
|
Not enough template
|
|
|
Reaction component concentrations not optimal
|
|
|
Reaction mix components are compromised
|
|
|
Primer design not optimal (causing
non-specific
annealing, or primer dimer formation)
|
|
|
Multiple/non-specific products from PCR reaction
|
Primer design not optimal (causing
non-specific
annealing, or primer dimer formation)
|
|
Template or reaction mixture components are contaminated
|
|
|
Annealing temperature too low
|
|
|
Primer concentration too high
|
|
|
Template concentration is not optimal
|