High GC PCR: Difference between revisions

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==<strong>How to: ''Streptomyces'' colony PCR</strong>==
==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==
#Streak your strain of interest for single colonies on [[DNA]].
#Streak your strain of interest for single colonies on [[DNA]].
#Pick a single colony and crush in 100;micro&l 50% DMSO.
#Pick a single colony and crush in 100;micro&l 50% DMSO.
#Heat at 65 degrees for 20 minutes.
#Heat at 65 degrees for 20 minutes.
#Add 1;micro&l of this to 10;micro&l of PCR mix
#Add 1;micro&l of this to 10;micro&l of PCR mix

Revision as of 16:04, 4 July 2019

High GC PCR

Actinobacteria typically have a high GC content in their DNA. High GC DNA templates can be difficult to amplify by PCR because of stronger hydrogen bonding and possible formation of secondary structures.

Tips for successful high GC PCR

  • Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity.
  • When designing primers, assess properties such as melting temperature and the likelihood of secondary structure formation. A useful tool for this is Net Primer (http://www.premierbiosoft.com/netprimer/).
  • When amplifying a region of DNA, try to find small regions of higher AT content that could form part of the primer sequence. This well help lower the Tm of the primers and reduce self-annealing and secondary structure formation.
  • Some specific polymerases can aid high GC DNA amplification. High fidelity polymerases will help improve specificity.
  • Q5® High-Fidelity DNA Polymerase comes with a high GC enhancer that can be added to reaction mixtures up to a final concentration of 10-20% to improve specificity.


General protocol for Streptomyces colony PCR

  1. Streak your strain of interest for single colonies on DNA.
  2. Pick a single colony and crush in 100;micro&l 50% DMSO.
  3. Heat at 65 degrees for 20 minutes.
  4. Add 1;micro&l of this to 10;micro&l of PCR mix