TECHVAULT > FAQ > APPLICATION SUPPORT > PCR AND SEQUENCING

My PCR reactions are not working, can you give me some trouble-shooting hints?

If you are not getting any amplification from any of these primers,  start by making sure  other reagents are working well by running the standard positive and negative controls.

dNTPs tend to go bad very quickly, particularly if repeatedly freeze and thaw them.  Check reagent concentrations (including the primers and template.)

If these are new sequences never used before, check for strong secondary structures which may affect the hybridization of the primers to the templates.

Try lowering your annealing temperature, increasing extension times, and adjusting your Mg concentration.  There are many variables in PCR which need to be monitored to get a successful reaction.  One particularly helpful website for setting up and troubleshooting PCR reactions is Tavi's website.

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