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class="content-sidebar-wrap"><main class="content"><article class="post-12907 post type-post status-publish format-standard has-post-thumbnail category-lab-tips-and-tricks tag-pcr-workflow entry" aria-label="PCR Primer Design Tips"><header class="entry-header"><h1 class="entry-title">PCR Primer Design Tips</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/behind-the-bench-staff/" title="Posts by Behind The Bench Staff" rel="author">Behind The Bench Staff</a> </span> <span class="post-info-date"> 09.25.2019 </span> </header><div class="entry-content"><p>Designing oligonucleotides and making sure that you have the right parameters for your <a href="https://www.thermofisher.com/blog/behindthebench/what-is-an-oligo/?icid=BID_SBOLG_PJT3762_WhatIsOligohttp://" target="_blank" rel="noopener noreferrer">oligo</a> is an important step in securing results, especially in <a href="https://www.thermofisher.com/us/en/home/life-science/oligonucleotides-primers-probes-genes/applied-biosystems-custom-primers-probes.html?icid=BID_SBOLG_PJT3762_PCRprimerDesign" target="_blank" rel="noopener noreferrer">PCR Primer Design</a>. In order to achieve successful DNA amplification, it’s important to start off with the right primer.</p> <p><img decoding="async" src="https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2019/09/pcr-primer.jpg" width="396" height="207" /></p> <h2>What makes a good primer?</h2> <p>Here are some guidelines for designing your <a href="https://www.thermofisher.com/us/en/home/life-science/oligonucleotides-primers-probes-genes/applied-biosystems-5-prime-labeled-primers.html?icid=BID_SBOLG_PJT3762_PCRprimers" target="_blank" rel="noopener noreferrer">PCR primers</a>:</p> <ol> <li>Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation.</li> <li>A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.</li> <li>Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other. Because the Tm is dependent on the length, it’s important to keep primers on the shorter end. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.</li> <li>Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting.</li> <li>Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT-rich domains.</li> <li>Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).</li> <li>Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.</li> <li>If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.</li> <li>If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.</li> <li>If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification.</li> </ol> <p> </p> <p>For Research Use Only. Not for Use in Diagnostic Procedures.</p> <aside class="mashsb-container mashsb-main "><div class="mashsb_above_buttons">Share this article </div><div class="mashsb-box"><div class="mashsb-count mash-large" style="float:left"><div class="counts mashsbcount">425</div><span class="mashsb-sharetext">SHARES</span></div><div class="mashsb-buttons"><a class="mashicon-facebook mash-large mashsb-noshadow" href="https://www.facebook.com/sharer.php?u=https%3A%2F%2Fwww.thermofisher.com%2Fblog%2Fbehindthebench%2Fpcr-primer-design-tips%2F" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Facebook</span></a><a class="mashicon-linkedin mash-large mashsb-noshadow" href="https://www.linkedin.com/shareArticle?trk=PCR+Primer+Design+Tips&url=https%3A%2F%2Fwww.thermofisher.com%2Fblog%2Fbehindthebench%2Fpcr-primer-design-tips%2F" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Linkedin</span></a><a class="mashicon-twitter mash-large mashsb-noshadow" href="https://twitter.com/intent/tweet?text=PCR%20Primer%20Design%20Tips&url=https://www.thermofisher.com/blog/behindthebench/?p=12907&via=thermofisher" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Twitter</span></a><a class="mashicon-mail mash-large mashsb-noshadow" href="mailto:?subject=Check%20out%20this%20article%20from%20Accelerating%20Science&body=Check%20out%20this%20article%20I%20just%20read%3Ahttps%3A%2F%2Fwww.thermofisher.com%2Fblog%2Fbehindthebench%2Fpcr-primer-design-tips%2F" target="_top" rel="nofollow"><span class="icon"></span><span class="text">Mail</span></a><div class="onoffswitch2 mash-large mashsb-noshadow" style="display:none"></div></div> </div> <div style="clear:both"></div><div class="mashsb-toggle-container"></div></aside> </div></article><div class="entry-comments" id="comments"><h3>Comments</h3><ol class="comment-list"> <li class="comment even thread-even depth-1" id="comment-7260"> <article id="article-comment-7260"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/a905c0855f5c6b396d9a3cd7ad128108?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/a905c0855f5c6b396d9a3cd7ad128108?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' decoding='async'/><span class="comment-author-name">Rijith Jayarajan</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-7260">April 18, 2020 at 10:26 pm</a></time></p> </header> <div class="comment-content"> <p>5. Try toavoidregionsofsecondarystructureandhaveabalanceddistributionofGC-rich and AT- rich domains.</p> <p>What is the 5th point??? </p> <p>Please edit properly before you upload something.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-7260' data-commentid="7260" data-postid="12907" data-belowelement="article-comment-7260" data-respondelement="respond" data-replyto="Reply to Rijith Jayarajan" aria-label='Reply to Rijith Jayarajan'>Reply</a></div> </article> <ul class="children"> <li class="comment odd alt depth-2" id="comment-7657"> <article id="article-comment-7657"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/37ee79e6445562dd76fb95287371bb39?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/37ee79e6445562dd76fb95287371bb39?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">YJ Loo</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-7657">May 25, 2020 at 5:00 pm</a></time></p> </header> <div class="comment-content"> <p>Try to avoid regions of secondary structure and have a balanced distribution of GC-rich and AT- rich domains.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-7657' data-commentid="7657" data-postid="12907" data-belowelement="article-comment-7657" data-respondelement="respond" data-replyto="Reply to YJ Loo" aria-label='Reply to YJ Loo'>Reply</a></div> </article> </li> </ul> </li> <li class="comment even thread-odd thread-alt depth-1" id="comment-7261"> <article id="article-comment-7261"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/a905c0855f5c6b396d9a3cd7ad128108?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/a905c0855f5c6b396d9a3cd7ad128108?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Rijith Jayarajan</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-7261">April 18, 2020 at 10:27 pm</a></time></p> </header> <div class="comment-content"> <p>5. Try toavoidregionsofsecondarystructureandhaveabalanceddistributionofGC-rich and AT-rich domains.</p> <p>What is the 5th point??? </p> <p>Please edit properly before you upload something.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-7261' data-commentid="7261" data-postid="12907" data-belowelement="article-comment-7261" data-respondelement="respond" data-replyto="Reply to Rijith Jayarajan" aria-label='Reply to Rijith Jayarajan'>Reply</a></div> </article> </li> <li class="comment odd alt thread-even depth-1" id="comment-13986"> <article id="article-comment-13986"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/420d11f6c9f96ced8a3132aa43300545?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/420d11f6c9f96ced8a3132aa43300545?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Nesrine</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-13986">December 1, 2020 at 11:27 am</a></time></p> </header> <div class="comment-content"> <p>4. Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting.<br /> What does this means please. Can I have an explanation !?</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-13986' data-commentid="13986" data-postid="12907" data-belowelement="article-comment-13986" data-respondelement="respond" data-replyto="Reply to Nesrine" aria-label='Reply to Nesrine'>Reply</a></div> </article> <ul class="children"> <li class="comment even depth-2" id="comment-14467"> <article id="article-comment-14467"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/394f802c313ba473ddf4721919a640e5?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/394f802c313ba473ddf4721919a640e5?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Assirbad Behura</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-14467">December 30, 2020 at 1:24 am</a></time></p> </header> <div class="comment-content"> <p>In case of designing cloning primers, 3 or 4 nucleotides are added at 5′ end before the restriction site to facilitate efficient cutting.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-14467' data-commentid="14467" data-postid="12907" data-belowelement="article-comment-14467" data-respondelement="respond" data-replyto="Reply to Assirbad Behura" aria-label='Reply to Assirbad Behura'>Reply</a></div> </article> </li> </ul> </li> <li class="comment odd alt thread-odd thread-alt depth-1" id="comment-13995"> <article id="article-comment-13995"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/88da37a8a02aedb7979abde160715fab?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/88da37a8a02aedb7979abde160715fab?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Niranjan</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-13995">December 2, 2020 at 4:37 am</a></time></p> </header> <div class="comment-content"> <p>Anyone please send the flow chart of primer design for Bacterial DNA diagnosis and Viral RNA diagnosis. Flow Chart means starting to end (Taking a sequence from NCBI to final primer design)</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-13995' data-commentid="13995" data-postid="12907" data-belowelement="article-comment-13995" data-respondelement="respond" data-replyto="Reply to Niranjan" aria-label='Reply to Niranjan'>Reply</a></div> </article> </li> <li class="comment even thread-even depth-1" id="comment-14468"> <article id="article-comment-14468"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/394f802c313ba473ddf4721919a640e5?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/394f802c313ba473ddf4721919a640e5?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Assirbad Behura</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-14468">December 30, 2020 at 1:27 am</a></time></p> </header> <div class="comment-content"> <p>In case of designing cloning primers, 3 or 4 nucleotides are added at 5′ end before the restriction site to facilitate efficient cutting.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-14468' data-commentid="14468" data-postid="12907" data-belowelement="article-comment-14468" data-respondelement="respond" data-replyto="Reply to Assirbad Behura" aria-label='Reply to Assirbad Behura'>Reply</a></div> </article> </li> <li class="comment odd alt thread-odd thread-alt depth-1" id="comment-14834"> <article id="article-comment-14834"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/b16a1a73bed54eda56c986b716d25812?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/b16a1a73bed54eda56c986b716d25812?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Ali Husain</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-14834">January 18, 2021 at 4:42 pm</a></time></p> </header> <div class="comment-content"> <p>Nice work, but please, can you explain point 6 “Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT)”??</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-14834' data-commentid="14834" data-postid="12907" data-belowelement="article-comment-14834" data-respondelement="respond" data-replyto="Reply to Ali Husain" aria-label='Reply to Ali Husain'>Reply</a></div> </article> <ul class="children"> <li class="comment even depth-2" id="comment-15411"> <article id="article-comment-15411"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/311ece51b07054e2e9dff21edb4b5461?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/311ece51b07054e2e9dff21edb4b5461?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Stephen Jackson</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-15411">March 18, 2021 at 3:02 pm</a></time></p> </header> <div class="comment-content"> <p>Oligos containing these types of sequences are typically more difficult to synthesize and may have non-Watson/Crick secondary structures that could interfere with their performance.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-15411' data-commentid="15411" data-postid="12907" data-belowelement="article-comment-15411" data-respondelement="respond" data-replyto="Reply to Stephen Jackson" aria-label='Reply to Stephen Jackson'>Reply</a></div> </article> </li> <li class="comment odd alt depth-2" id="comment-15413"> <article id="article-comment-15413"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/311ece51b07054e2e9dff21edb4b5461?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/311ece51b07054e2e9dff21edb4b5461?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Steve Jackson</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-15413">March 18, 2021 at 3:04 pm</a></time></p> </header> <div class="comment-content"> <p>Oligos containing these types of sequences are typically more difficult to synthesize and may demonstrate non-Watson/Crick secondary structures, changing their behavior.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-15413' data-commentid="15413" data-postid="12907" data-belowelement="article-comment-15413" data-respondelement="respond" data-replyto="Reply to Steve Jackson" aria-label='Reply to Steve Jackson'>Reply</a></div> </article> </li> </ul> </li> <li class="comment even thread-even depth-1" id="comment-15412"> <article id="article-comment-15412"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/311ece51b07054e2e9dff21edb4b5461?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/311ece51b07054e2e9dff21edb4b5461?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Stephen Jackson</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-15412">March 18, 2021 at 3:02 pm</a></time></p> </header> <div class="comment-content"> <p>Oligos containing these types of sequences are typically more difficult to synthesize and may have non-Watson/Crick secondary structures that could interfere with their performance.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-15412' data-commentid="15412" data-postid="12907" data-belowelement="article-comment-15412" data-respondelement="respond" data-replyto="Reply to Stephen Jackson" aria-label='Reply to Stephen Jackson'>Reply</a></div> </article> </li> <li class="comment odd alt thread-odd thread-alt depth-1" id="comment-15448"> <article id="article-comment-15448"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/53c10e759b25fb6d557b13c4d1f56aa7?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/53c10e759b25fb6d557b13c4d1f56aa7?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">mustafa younis</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-15448">March 22, 2021 at 6:16 pm</a></time></p> </header> <div class="comment-content"> <p>could any one help me with determining what set of primers go with these PCR reactions:<br /> Set 1: 5’TACACTTATACTTTC3’ and 3’GTAAACCGCGCATTAG5’<br /> Set 2: 5’CTCGAGGTGAATAT3’ and 3’CCGCGCATTAGCTAT5’<br /> Set 3: 5’GAGTTACACTTATAC3’ and 3’TGGCGAGTAATCGATA5’</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-15448' data-commentid="15448" data-postid="12907" data-belowelement="article-comment-15448" data-respondelement="respond" data-replyto="Reply to mustafa younis" aria-label='Reply to mustafa younis'>Reply</a></div> </article> <ul class="children"> <li class="comment even depth-2" id="comment-15483"> <article id="article-comment-15483"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/dc8610a411313dd2c4b5007e16031a23?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/dc8610a411313dd2c4b5007e16031a23?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Alex</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-15483">March 26, 2021 at 3:30 am</a></time></p> </header> <div class="comment-content"> <p>those already look like primers to me, unless you’re doing 6 PCR reactions</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-15483' data-commentid="15483" data-postid="12907" data-belowelement="article-comment-15483" data-respondelement="respond" data-replyto="Reply to Alex" aria-label='Reply to Alex'>Reply</a></div> </article> </li> </ul> </li> <li class="comment odd alt thread-even depth-1" id="comment-15852"> <article id="article-comment-15852"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/ad146fa169bbdb75f6397aade06f160f?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/ad146fa169bbdb75f6397aade06f160f?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">YJ</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-15852">May 4, 2021 at 8:31 pm</a></time></p> </header> <div class="comment-content"> <p>Great info! But I was wondering how primers can be designed in such a way that one of them is synthesised first?</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-15852' data-commentid="15852" data-postid="12907" data-belowelement="article-comment-15852" data-respondelement="respond" data-replyto="Reply to YJ" aria-label='Reply to YJ'>Reply</a></div> </article> </li> <li class="comment even thread-odd thread-alt depth-1" id="comment-15996"> <article id="article-comment-15996"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/43784fe70f829e52519bd7ede27ed704?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/43784fe70f829e52519bd7ede27ed704?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">LM</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-15996">May 17, 2021 at 5:40 pm</a></time></p> </header> <div class="comment-content"> <p>I have a confusion. Can designing a pcr reaction and designing of PCR primers are equal?</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-15996' data-commentid="15996" data-postid="12907" data-belowelement="article-comment-15996" data-respondelement="respond" data-replyto="Reply to LM" aria-label='Reply to LM'>Reply</a></div> </article> <ul class="children"> <li class="comment odd alt depth-2" id="comment-16798"> <article id="article-comment-16798"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/8342967568300fee3910fe31d828bb7c?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/8342967568300fee3910fe31d828bb7c?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Abel</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-16798">July 16, 2021 at 10:18 pm</a></time></p> </header> <div class="comment-content"> <p>PCR primers are just one of the “ingredients” for PCR. Besides primers you will need a polymerase, nucleotides, minerals, ex. and those details, along with the thermocycling temperatures and number of cycles, are what constitute the design of the PCR reaction. There are plenty of protocols you can find online of how to design a PCR reaction based on what you are hoping to accomplish.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-16798' data-commentid="16798" data-postid="12907" data-belowelement="article-comment-16798" data-respondelement="respond" data-replyto="Reply to Abel" aria-label='Reply to Abel'>Reply</a></div> </article> </li> </ul> </li> <li class="comment even thread-even depth-1" id="comment-16666"> <article id="article-comment-16666"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/dc28901b515d7fad44501d9a87397f0a?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/dc28901b515d7fad44501d9a87397f0a?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">messa</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-16666">June 30, 2021 at 3:55 pm</a></time></p> </header> <div class="comment-content"> <p>which ends of primer should have more CG cnontent? and why?</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-16666' data-commentid="16666" data-postid="12907" data-belowelement="article-comment-16666" data-respondelement="respond" data-replyto="Reply to messa" aria-label='Reply to messa'>Reply</a></div> </article> <ul class="children"> <li class="comment odd alt depth-2" id="comment-25527"> <article id="article-comment-25527"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/7bb158af658c980adea966ef21ae5217?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/7bb158af658c980adea966ef21ae5217?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">emmanuel</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-25527">March 22, 2023 at 4:13 pm</a></time></p> </header> <div class="comment-content"> <p>why do we consider start, stop, melting temperature, gc content, primer length, template strand: plus minus. in designing a pcr primer</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-25527' data-commentid="25527" data-postid="12907" data-belowelement="article-comment-25527" data-respondelement="respond" data-replyto="Reply to emmanuel" aria-label='Reply to emmanuel'>Reply</a></div> </article> </li> </ul> </li> <li class="comment even thread-odd thread-alt depth-1" id="comment-17612"> <article id="article-comment-17612"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/b4db85500a44ed1cccc13e8021837fa5?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/b4db85500a44ed1cccc13e8021837fa5?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Astrid</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-17612">September 9, 2021 at 1:45 am</a></time></p> </header> <div class="comment-content"> <p>If i want to design primers at the program..<br /> this is the DNA sequence that i want to PCR..</p> <p>5’GAAGAAAGAA CACAAAAAGC AGGAAGGGAA GGGGACACAG<br /> CCAGGTTTGA TGAGCCCAGA CTCTCCATTA AACGCTGCGC<br /> TCAGGGGCCC CAGCTGGGGT CATTGACCCC TGAGCAACAC<br /> TCTCTTAAGC TGGAGGATCT CGCCTTGCAG GTTGAAGAGG<br /> TTACAACACA GAGCAGGGGC TGGGGCCTGG CATGAGGCAC<br /> 3’TGATCAATCA GTGCCACAGA TACTGCTGTT CTTAGGATGA<br /> GAGCAGAAGC TGGGGTGTCC CACCACAAGC AGGACAGAGA<br /> GGACACCTCT TTGTCATCAG AGATTAATGG GACCCTTGTC<br /> AGGCCCAGGG TGCAGTGGGG GAAGGGCAGA AGGGACCCTG<br /> CCCTCTCAAA GGCACAGTGA AGCTCTGAAC TGAAGGAGCT </p> <p>Should I used sequence from 5′ end?<br /> and can i check if the primers resulting the correct sequence?</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-17612' data-commentid="17612" data-postid="12907" data-belowelement="article-comment-17612" data-respondelement="respond" data-replyto="Reply to Astrid" aria-label='Reply to Astrid'>Reply</a></div> </article> </li> <li class="comment odd alt thread-even depth-1" id="comment-17911"> <article id="article-comment-17911"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/7dd2dfeb4cf9cee9aa1f17c7aa526848?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/7dd2dfeb4cf9cee9aa1f17c7aa526848?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Emma Smith</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-17911">October 8, 2021 at 8:39 pm</a></time></p> </header> <div class="comment-content"> <p>Hello,</p> <p>I need this site for guest posting.This time my budget is low. So I can pay you 5$ each post. So I accepted my offer and I sent you an article.</p> <p>I have some questions ? can you please answer me. If you do or not with price.</p> <p>1st : You accepted a casino gambling adult link on your site ?</p> <p>2nd ; How much you can charge to insert a link ?</p> <p>3rd : How many visitors come to your site in one month ?</p> <p>4rth ; How many word article are accepted on your site ?</p> <p>Waiting for your reply with a positive response.</p> <p>Thanks.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-17911' data-commentid="17911" data-postid="12907" data-belowelement="article-comment-17911" data-respondelement="respond" data-replyto="Reply to Emma Smith" aria-label='Reply to Emma Smith'>Reply</a></div> </article> </li> <li class="comment even thread-odd thread-alt depth-1" id="comment-19578"> <article id="article-comment-19578"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/d489cab1b5c2a56863f15bc0effca454?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/d489cab1b5c2a56863f15bc0effca454?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Manish</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-19578">December 30, 2021 at 7:15 am</a></time></p> </header> <div class="comment-content"> <p>Hi,<br /> My Primer length is 42 nucleotides, (main seq. is 30nts, rest is adapters and Recognition seq.)<br /> GC content is~33, Tm= 64, HPLC purified.<br /> Primer seq.- GCGG TTAATTAA ATG GCA AAT TAT AAT TTC AAG TAC ATC GCC<br /> I am not getting any amplification product.<br /> Suggestions please!!</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-19578' data-commentid="19578" data-postid="12907" data-belowelement="article-comment-19578" data-respondelement="respond" data-replyto="Reply to Manish" aria-label='Reply to Manish'>Reply</a></div> </article> </li> <li class="comment odd alt thread-even depth-1" id="comment-19579"> <article id="article-comment-19579"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/d489cab1b5c2a56863f15bc0effca454?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/d489cab1b5c2a56863f15bc0effca454?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Manish</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-19579">December 30, 2021 at 7:19 am</a></time></p> </header> <div class="comment-content"> <p>Hi,<br /> My primer length is 42 nts. (main seq.= 30 nts, rest is adapters and Recognition seq.)<br /> GC content= 33, Tm= 64, HPLC purified<br /> Primer seq.- GCGG TTAATTAA ATG GCA AAT TAT AAT TTC AAG TAC ATC GCC<br /> I am not getting gene specific amplification product (probably, primer dimer only)<br /> Suggestions please!!</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-19579' data-commentid="19579" data-postid="12907" data-belowelement="article-comment-19579" data-respondelement="respond" data-replyto="Reply to Manish" aria-label='Reply to Manish'>Reply</a></div> </article> </li> <li class="comment even thread-odd thread-alt depth-1" id="comment-19580"> <article id="article-comment-19580"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/d489cab1b5c2a56863f15bc0effca454?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/d489cab1b5c2a56863f15bc0effca454?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Manish</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-19580">December 30, 2021 at 7:26 am</a></time></p> </header> <div class="comment-content"> <p>Hi,<br /> My primer length is 42 nts. (Main seq.30 nts, Rest is adapters and recognition seq.)<br /> GC= 33, Tm=64°C, HPLC purified.<br /> I am not getting my gene specific product (probably, primer dimer only)<br /> Primer seq.= GCGG TTAATTAA ATG GCA AAT TAT AAT TTC AAG TAC ATC GCC<br /> Suggestions please !!</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-19580' data-commentid="19580" data-postid="12907" data-belowelement="article-comment-19580" data-respondelement="respond" data-replyto="Reply to Manish" aria-label='Reply to Manish'>Reply</a></div> </article> </li> <li class="comment odd alt thread-even depth-1" id="comment-20775"> <article id="article-comment-20775"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/8a2423692773edcd9fab22c0c27724b4?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/8a2423692773edcd9fab22c0c27724b4?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Inell Poullard</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-20775">May 26, 2022 at 9:00 pm</a></time></p> </header> <div class="comment-content"> <p>Napisy na blogu sa zazwyczaj pokazywane jak cechy elektroniczne w przeciwnej kolejnosci chronologicznej, z najrózniejszym wpisem pojawiajacym sie u góry strony. Posty zazwyczaj zawieraja tekst, obrazy, filmy, wykresy wiadomosci a wykresy. Tresc zapisu na blogu jest przewaznie napisana w trendzie narracyjnym, który rozbija sie osobistymi doswiadczeniami czy opiniami na okreslony temat.</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-20775' data-commentid="20775" data-postid="12907" data-belowelement="article-comment-20775" data-respondelement="respond" data-replyto="Reply to Inell Poullard" aria-label='Reply to Inell Poullard'>Reply</a></div> </article> </li> <li class="comment even thread-odd thread-alt depth-1" id="comment-21901"> <article id="article-comment-21901"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/f8d5f04ca517c0cc222c6193ed091522?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/f8d5f04ca517c0cc222c6193ed091522?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Petrick zag</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-21901">August 29, 2022 at 7:15 am</a></time></p> </header> <div class="comment-content"> <p>Really nice content about pcr primer designer tips.Thank You for sharing such a nice post<br /> ,thanks for sharing. Good stuff!</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-21901' data-commentid="21901" data-postid="12907" data-belowelement="article-comment-21901" data-respondelement="respond" data-replyto="Reply to Petrick zag" aria-label='Reply to Petrick zag'>Reply</a></div> </article> </li> <li class="comment odd alt thread-even depth-1" id="comment-24114"> <article id="article-comment-24114"> <header class="comment-header"> <p class="comment-author"> <img alt='' src='httpss://secure.gravatar.com/avatar/a57f8377c862211091931f2efba21a0a?s=48&d=mm&r=g' srcset='httpss://secure.gravatar.com/avatar/a57f8377c862211091931f2efba21a0a?s=96&d=mm&r=g 2x' class='avatar avatar-48 photo' height='48' width='48' loading='lazy' decoding='async'/><span class="comment-author-name">Hasan Babar</span> <span class="says">says</span> </p> <p class="comment-meta"><time class="comment-time"><a class="comment-time-link" href="https://www.thermofisher.com/blog/behindthebench/pcr-primer-design-tips/#comment-24114">December 20, 2022 at 5:41 pm</a></time></p> </header> <div class="comment-content"> <p>Nice job</p> </div> <div class="comment-reply"><a rel='nofollow' class='comment-reply-link' href='#comment-24114' data-commentid="24114" data-postid="12907" data-belowelement="article-comment-24114" data-respondelement="respond" data-replyto="Reply to Hasan Babar" aria-label='Reply to Hasan Babar'>Reply</a></div> </article> </li> </ol></div> <div id="respond" class="comment-respond"> <h3 id="reply-title" class="comment-reply-title">Leave a Reply <small><a rel="nofollow" id="cancel-comment-reply-link" href="/behindthebench/pcr-primer-design-tips/#respond" style="display:none;">Cancel reply</a></small></h3><form action="https://www.thermofisher.com/blog/behindthebench/wp-comments-post.php" method="post" id="commentform" class="comment-form" novalidate><p class="comment-notes"><span id="email-notes">Your email address will not be published.</span> <span class="required-field-message">Required fields are marked <span class="required">*</span></span></p><p class="comment-form-comment"><label for="comment">Comment <span class="required">*</span></label> <textarea id="comment" name="comment" cols="45" rows="8" maxlength="65525" required></textarea></p><p class="comment-form-author"><label for="author">Name <span class="required">*</span></label> <input id="author" name="author" type="text" value="" size="30" maxlength="245" autocomplete="name" required /></p> <p class="comment-form-email"><label for="email">Email <span class="required">*</span></label> <input id="email" name="email" type="email" value="" size="30" maxlength="100" aria-describedby="email-notes" autocomplete="email" required /></p> <p class="comment-form-url"><label for="url">Website</label> <input id="url" name="url" type="url" value="" size="30" maxlength="200" autocomplete="url" /></p> <p class="form-submit"><input name="submit" type="submit" id="submit" class="submit" value="Post Comment" /> <input type='hidden' name='comment_post_ID' value='12907' id='comment_post_ID' /> <input type='hidden' name='comment_parent' id='comment_parent' value='0' /> </p><p style="display: none;"><input type="hidden" id="akismet_comment_nonce" name="akismet_comment_nonce" value="0135c5ebd8" /></p><p style="display: none !important;" class="akismet-fields-container" data-prefix="ak_"><label>Δ<textarea name="ak_hp_textarea" cols="45" rows="8" maxlength="100"></textarea></label><input type="hidden" id="ak_js_2" name="ak_js" value="118"/><script>document.getElementById( "ak_js_2" ).setAttribute( "value", ( new Date() ).getTime() );</script></p></form> </div> <div class="adjacent-entry-pagination pagination"><div class="pagination-previous alignleft"><i class="fa fa-icon fa-chevron-left"></i> <a href="https://www.thermofisher.com/blog/behindthebench/improved-understanding-of-fetal-abnormalities-with-cytoscan-xon-and-exome-sequencing/" rel="prev">Improved Understanding of Fetal Abnormalities with CytoScan XON and Exome Sequencing</a></div><div class="pagination-next alignright"><a href="https://www.thermofisher.com/blog/behindthebench/precision-prescribing-with-pharmacogenomics/" rel="next">Precision Prescribing with Pharmacogenomics</a> <i class="fa fa-icon fa-chevron-right"></i></div></div></main></div></div><footer class="site-footer"><div class="wrap"><div class="creds"><p class="footer-links"><a href="https://corporate.thermofisher.com/en/footer/privacy-statement.html" target="_blank" rel="noopener noreferrer">Privacy Statement</a><a href="https://corporate.thermofisher.com/en/footer/terms-and-conditions.html" target="_blank" rel="noopener noreferrer">Terms & Conditions</a><a href="/blog/locations/">Locations</a><a href="/blog/sitemap/">Sitemap</a></p><p>© 2024 <a href="https://www.thermofisher.com">Thermo Fisher Scientific</a>. 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