How to Design Primers for PCR

Are you looking to design a primer for your PCR? Jennifer Tsang, Science Communication and Marketing Coordinator at Addgene, is here with some tips for creating successful primers. For the full text protocol, visit addgene.org/protocols/primer-design/

Oligonucleotide primers are necessary when running a PCR reaction. One needs to design primers that are complementary to the template region of DNA. They are synthesized chemically by joining nucleotides together. One must selectively block and unblock repeatedly the reactive groups on a nucleotide when adding a nucleotide one at a time. The main property of primers is that they must correspond to sequences on the template molecule (must be complementary to template strand). However, primers do not need to correspond to the template strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the template DNA strand so elongation can proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches of several nucleotides. Also, both of the 3’ ends of the hybridized primers must point toward one another.
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Chapters:
0:00 Introduction to Primer Design
0:57 Primer Length
1:26 Annealing & Melting Temperatures
2:27 Makeup of Nucleotides in the Primer
3:20 Secondary Structure
4:04 You Just Designed a Primer!

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Credits
Featuring: Jennifer Tsang
Shot by: Jennifer Tsang
Written by: Jennifer Tsang & Quintin Marcelino
Directed, Animated, & Edited by: Quintin Marcelino
Music by: Lauren Duski, Vibe Mountain, & RKCV