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Real-Time PCR: General Considerations, Rev A

anscription. It is also helpful to design primers at splice junctions to avoid genomic DNA amplification

Template and Primer Design
Template Design
A successful real-time PCR reaction requires efficient amplification of the product. Both primers and target sequence affect this efficiency. Significant template secondary structure may hinder the primers from annealing and prevent complete product extension by the polymerase. Follow these guidelines:
Amplify a template region of 75150 bp
Avoid secondary structure if possible
Use an annealing temperature above the melting temperature (Tm) for any template secondary structures
Avoid templates with long (>4) repeats of single bases
Maintain a GC content of 5060%
Analyze secondary structure with the DNA mfold server of Dr. Michael Zuker or equivalent program at:

Secondary Structure Analysis
To evaluate secondary structure of a product on Dr. Zukers site:
1. Name the sequence example: bactin1
2. Copy the sequence or retype it into the big text box
3. Scroll down the page to the input labeled Folding temperature and enter the annealing temperature of the reaction
4. Scroll down the page to the input labeled Ionic conditions and change the units to mM. Adjust the values to reflect the ionic conditions in the reaction (set [Na+] to 50 mM and [Mg2+] to 3 mM for most reactions)
5. Scroll down further to enter your e-mail address you will not receive an e-mail message,


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