Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

Table of Contents

Expand All | Contract All

Designing PCR Primers Using Primer3Plus

(Protocol summary only for purposes of this preview site)

Designing oligonucleotide primers is a crucial step for successful molecular biology experiments that require the use of PCR. PCR involves cycles of three steps: denaturation, annealing, and extension. During denaturation, double-stranded DNA (dsDNA) molecules (templates) are separated into single strands. During annealing, a pair of primers is annealed to the complementary regions of the single-stranded molecules. In the extension step, DNA polymerase extends the primers to produce DNA molecules that correspond to the region bracketed by the primers (the amplicons). All of these steps are temperature sensitive, and the common choice of temperatures is 94C, 60C, and 70C, respectively. Poorly designed primers may lead to no amplification product or additional undesired amplified fragments. The goals of primer design include good primer specificity, high annealing efficiency, appropriate melting temperature, proper GC content, and the prevention of primer hairpins or primer dimers (Burpo 2001), which are discussed in detail in the Discussion section of this protocol and in Chapter 7.


Previous   |   Next

Save 30% & Get Free Shipping!

Save 30% at checkout on our website.
(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

Search for information about other protocols included in the book:

Read What Others Are Saying About Molecular Cloning:

Tom Maniatis wins Lasker

'Not Quite a Book Prize'

Get alerts about special offers from Cold Spring Harbor Laboratory Press free to your Inbox