Primer Design

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Table of Contents
Primer Design 2 What to Avoid When Designing Primers for PCR 16
• Repeats/Runs 16
1. The Science Behind Primers 3
• Cross homology 16
What are primers? 3
• Primer Secondary Structure and Gibbs Free Energy 16
• W
 hat types of primers are there?
• Template secondary structure 19
RNA primers vs. DNA primers 3
Primers in DNA replication 4
3. In Silico Primer Design Tools 19
• Forward and Reverse Primers 5
Desktop vs. Cloud-Based Primer Design Tools 19
Primers in DNA Amplification and Scientific Applications 6
• Primer3 and Primer3Plus 20
• Primer Design for Cloning 7
• NCBI Blast 20
- Primer Design for Digest and Ligate Cloning Method 8
- Primer Design for the Golden Gate Cloning Method 9 4. Benchling’s Molecular Biology Tools 21
- Primer Design for the Gibson Cloning Method 10 Design Primers with Benchling 22
• Manual Primer Design 23
2. Primer Design for PCR 11
• Wizard Primer Design 26
Best Practices for PCR Primer Design 13
• Search, Import, and Attach Existing Primers Easily 27
• Primer Length 13
• Send Primers to NCBI Blast 30
• Primer Melting Temperature (Tm) 13
• Build Custom Primer Libraries 30
• Primer Annealing temperature (Ta) 14
• In silico PCR within Benchling 31
• Primer GC Content and GC Clamp 15
• Free Primer Design Tools for Academics 32
• Set Restriction Enzyme Cut Sites 15
• Request a Demo of Benchling 33
• End Stability 15
Benchling Primer Design and Molecular Biology Resources 34

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Primer Design
Primers are key ingredients in DNA synthesis, a process that occurs
in sequencing, cloning, PCR, and other molecular biology methods
in the lab. With Benchling, teams can easily access shared primer
libraries, upload new primer sequences, or design brand new primers.
Link primer information directly in the Benchling Notebook and
Benchling Registry providing full traceability for every experiment
where a primer was used. Be able to easily attribute results from
experiments with the exact set of primers used, or see which
sequences a primer is associated with. Once primers are designed,
run in silico PCR, or use them to plan critical tasks such as restriction
cloning, Golden Gate assembly, and Gibson cloning.

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Section 1 The Science Behind Primers

1.1 What are primers?

Primers are simple but key ingredients for DNA synthesis both within our bodies and within scientific
experiments. Primers can also be called oligonucleotides and are literally small pieces of single-
stranded nucleotides, generally about 5 - 22 base pairs in length. The main property of primers is
they must be complementary to the DNA template strand, serving to “prime” the strand for DNA
polymerase to bind to and initiate DNA synthesis.

What types of primers DNA Primers RNA Primers

are there? RNA primers
Use In vitro: PCR amplification, DNA In vivo: DNA replication
vs. DNA primers sequencing, cloning, and more

Reaction Amplification is Replication is enzyme-

Living organisms solely use RNA temperature-dependent, dependent catalytic reaction,
primers, while primers used in the lab requiring fewer proteins requiring several proteins
are usually DNA primers. Scientists use
Length 18 - 24 base pairs 10 - 20 base pairs
DNA primers instead of RNA primers
for a variety or reasons. DNA primers Creation Chemically synthesized by Primase (a type of RNA
scientists polymerase)
are far more stable and easier to store,
and they require less hard-to-come- Viability Longer-lived, more stable Shorter-lived, more reactive
by enzymes to initiate synthesis (see
Figure 1).

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 The binding of DNA or RNA primers to the template strand initiates the enzyme responsible for DNA
synthesis, DNA polymerase, to begin adding nucleotides to the reactive 3’-hydroxyl end (called the
“3 prime end”) of a existing nucleic acid on the primer, elongating and replicating the parent strand.

1.2 Primers in DNA replication

It is the role RNA primers play in DNA synthesis that makes primer biology so incredibly important.
In vivo, this is DNA replication or gene synthesis, a cornerstone of heredity where genetic material
that was inherited from parent cells is copied for cell growth, division, and differentiation.

DNA replication begins at an origin of replication, which is identified by proteins called initiators
that latch and make small openings in the double-stranded DNA double (dsDNA) helix. At these
breaks, helicase enzymes break the bonds between the double strands, exposing single-stranded
DNA (ssDNA) in a Y-shaped end called the DNA replication fork. The replication fork is where DNA
replication will actually occur, providing two strands of parent DNA now exposed to act as the DNA
template (gray strands below).

Figure 1. The process and components of DNA replication

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Next, RNA primase lays down the RNA primer on each of the parent template strands, providing
the 3’ end for a new polynucleotide strand to begin forming. DNA polymerase binds and synthesis
is initiated, with daughter strands being formed (purple strands). In this diagram, the top daughter
strand is called the leading strand and is exposed in the 5 prime (5’) → 3’ prime (3’) direction right to
left, allowing DNA synthesis to occur continuously as the helix unwinds.

Lagging strand synthesis of the bottom daughter strand occurs in the 5’ → 3’ direction left to right,
where DNA synthesis must continually start over as the helix unwinds. This creates many short
fragments of synthesized products, known as Okazaki fragments which average ~150-200 nucleotides
long. Each Okazaki fragment contains an RNA-DNA joint as a consequence of RNA priming, a
discovery that was important evidence for determining the role of RNA primers in DNA replication.
Okazaki fragments must be joined together by the enzyme ligase, which catalyzes the formation of
covalent phosphodiester bonds between two strands of DNA, ligating them together.

Forward and Reverse Primers

5’ 3’ 5’ 3’
Primers only bind to single-stranded DNA.
5’ 3’
In DNA replication in vivo, this is made 3’ 5’ 3’ 5’
available at the replication fork. In vitro, 5’ 3’
Reverse primer
scientists can create ssDNA by denaturing
Forward primer
dsDNA with heat, effectively breaking 3’ 5’
5’ 3’ 5’ 3’
hydrogen bonds that hold the two strands 3’ 5’

together. This creates two linear template 3’ 5’ 3’ 5’

strands where DNA synthesis can occur
continuously on both strands, often called
the top and bottom strand in place of the
Figure 2. Forward and reverse primers are complimentary strands of DNA that bind
terms leading and lagging strands.
at specific loci on single stranded DNA at the replication fork. The 5’ end of the primer
attaches to the 3’ end of the target DNA strand.

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 A pair of primers must be used, one for the top strand and one for the bottom. The primer pair binds
on opposite ends of the sequence being amplified, with their 3’ ends pointing toward each other.
The oligonucleotide primers being designed do not actually have to hybridize completely to the
template strand; it is essential, however, that the 3’ end corresponds completely to the template
DNA for elongation to occur. The forward primer anneals to the bottom strand that runs 3’ to 5’, and
the reverse primer anneals to the complementary bottom strand of DNA that runs 5’ to 3’. After
elongation, this results in two new strands of double-stranded DNA: one made from the forward
primer; and the other made from the reverse primer.

In order for the primers to bind to the template strand, it’s important they are not complementary to
each other. Primers complementary to each other can result in the creation of primer dimers, which
are two primers binding to each other instead of the template strand. Primer dimers are discussed in
detail in the section on what to avoid when designing primers for PCR.

1.3 Primers in DNA Amplification and

Scientific Applications
DNA primers have been developed for use in molecular biology methods
such as the ubiquitous polymerase chain reaction (PCR). Tips for how to
design primers for PCR is the main focus in this article and is discussed
in the section 2. However, primers are also used in DNA sequencing and
recombinant DNA technology methods such as restriction cloning and
newer DNA assembly methods such as Golden Gate and Gibson DNA
assembly methods — all of which are also offered in Benchling.

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Primer Design for Cloning
Molecular cloning is an important technique that allows scientists to replicate specific target
segments of DNA in host cells. The target gene or fragment of interest can be inserted into a cloning
vector, generally a circular prokaryotic plasmid genome (sometimes simply called a plasmid vector),
which can then be transformed and taken up by a specific recipient cell line, usually a bacterial cell.
The vector will then be incorporated into the host genome via DNA recombination, and as the cell line
naturally undergoes DNA replication as outlined above, the target gene of interest will also replicate,
or begin being cloned.

But first scientists must isolate the specific target

gene to be cloned. They can do this by using PCR
to amplify the target gene if it’s already isolated.
Alternatively, if the desired sequence to be amplified
is in a plasmid or in another sequence, they can find
restriction sites flanking the sequence in order to
cut it out using restriction enzymes, or rather, digest
the sequence. Both methods allow scientists to
produce a specific stretch of DNA that they can then
assemble into a plasmid vector to be transformed
into cell lines for cloning.

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Primer Design for Digest and Ligate Cloning Method
Common restriction enzymes (also called restriction endonucleases) such as EcoR1 recognize
and cut at specific palindromic restriction sites in the plasmid. EcoR1 cuts in a zig-zag across the
double-stranded DNA creating sticky ends, or single-stranded DNA overhangs of a few base pairs
long. Other enzymes like Alu1 are blunt end restriction enzymes that create blunt ends without
overhangs. Generally, the same restriction enzymes that cut the plasmid are also used to digest the
gene of interest out of its parent DNA, creating complementary sticky ends in the plasmid and target
sequence. Once the two are combined in solution, the target fragment can ligate into the plasmid
through hydrogen bonding. Gaps are again filled by DNA ligase and the plasmid is ready to be used in
further experiments, such as transformation.

This is considered the traditional digest and ligate method that will ultimately create recombinant
DNA. Recombinant DNA is formed in the laboratory by combining genetic material from multiple
sources, including DNA material from two different species.

EcoR1
recognition
sequence
GA
AT T

Ligate
A AG

C
T T

Cleavage by EcoR1 Assemble

C

Gene of
Plasmid with interest Create Recombinant
restriction site sticky ends plasmid DNA

Figure 3. The process of digestion and ligation cleaves a plasmid at a specific site
and adds a gene of interest into the plasmid.

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Another common alternative is to design primers with restriction sites at their 5’ ends. After PCR, this
creates a copy of the target DNA region with restriction sites needed at the end of the fragment. This
method is used when there may not be restriction sites for the same enzymes in the correct place in
both the vector and insert.

Traditional digest and ligate methods limit scientists to a single insert per cloning cycle. Advances in
cloning technologies have focused more on the assembly of multiple DNA fragments, which is made
much more efficient with assembly techniques such as Golden Gate cloning and Gibson cloning.

Primer Design for the Golden Gate Cloning Method

Also known as Golden Gate assembly, this cloning method allows scientists to simultaneously
assemble a vector (the backbone, usually a plasmid) with one or multiple DNA insert fragments
(the parts) into a single construct. Golden Gate uses Type IIs restriction enzymes, which unlike the
standard Type II enzymes, cut outside of their recognition sites, creating non-palindromic sticky end
overhangs. This allows for multiple fragments of DNA to be assembled by using combinations of
overhang sequences on the insert fragments that allow adjacent fragments to easily anneal together.
The Type IIs restriction enzymes allow for Golden Gate assembly to be considered scarless, meaning it
leaves no restriction sites left over after cloning.

Designing primers for Golden Gate cloning is automatic with Benchling’s primer design and DNA
assembly tools. For Golden Gate, the PCR primers should overlap adjacent DNA fragments to include
restriction sites and be designed in a way that when digested with a Type IIS enzyme, directional
assembly of the fragments is possible with DNA ligase.

Benchling’s Golden Gate Assembly wizard tool was built in conjunction with scientists from New
England Biolabs. The assembly wizard helps to automate the time-consuming and error-prone

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process of selecting appropriate sticky ends, while also supporting more complex techniques like site-
directed mutagenesis and the integration of spacers between fragments.

Primer Design for the Gibson Cloning Method

Also known as Gibson assembly and created
by Daniel G. Gibson in 1996, the Gibson cloning
protocol is similar to the Golden Gate method
in that it is scarless and can simultaneously
assemble a backbone sequence with multiple
DNA fragments into a single construct. What’s
unique about Gibson cloning is that it relies
on recombination rather than restriction
digestion and ligation to generate plasmids.

Gibson assembly requires scientists to produce

identical homologous overlaps at the ends
~20-40 base pairs long on both the target
DNA fragments to be assembled, and on each
side of the linearized vector. This creates an
identical overlap that can be chewed back
by exonuclease, leaving complementary
overhangs to assist in assembly.

Figure 4. Gibson assembly takes two pieces of DNA with homologous,

overlapping ends, removes the 5’ end of each overlap, joins, or anneals,
the overhanging strands of DNA, and fuses them together.

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Section 2 Primer Design for PCR

The polymerase chain reaction (PCR) is one of the most important methods in molecular biology.
Millions or billions of copies of a selected gene or specific DNA fragment can be created in a few
hours from a small sample and simple ingredients. The is known as PCR amplification, or gene
amplification. These millions or billions of DNA fragments are called PCR products and can be used
in various ways post-amplification. If you need an in-depth reminder on how PCR works, please see
this video review.

The main components of PCR are dsDNA, free nucleotides, a custom designed PCR primer pair,
a heat stable Taq polymerase, and a thermocycler. A typical PCR reaction involves mixing these
ingredients in a test tube with buffers and putting them through repeated cycles of heating and
cooling with a thermocycler.

Denaturing (95°C) Annealing (55 - 65°C) Extension (72°C)

Heating separates, or denatures, Cooling allows primers to bind to Raising the temperature slightly allows
dsDNA into ssDNA. the complementary sequence on Taq polymerase to bind to the 3’ primer
the single-stranded DNA template, end and begin synthesizing new strands of
known as primer annealing. DNA, known as primer extension.

These steps are repeated for 25-35 cycles. Every new DNA that is synthesized from the round before
can serve as a template for the next round, creating exponential growth of the target DNA. If a high
quality, purified DNA template was used and there were no issues with PCR reaction, the target region
can go from a single template to several copies to upwards of billions.

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Figure 5. Designing primers for PCR requires DNA primer pairs, free nucleotides, and target DNA. Step 1.
Denaturation separates the two strands of DNA Step 2. Primers join, or anneal, to the individual strands of
the target DNA Step 3. Primer DNA is extended at the primer’s 3’ end. Steps 1-3 are then repeated.

Designing primers for PCR is essential for successful PCR reactions and initiating DNA amplification.
PCR primers must be custom designed to be complementary to the template region of DNA and
must code for only the specific upstream and downstream DNA sites of the sequence being
amplified. Therefore, it’s key that sufficient sequence information is known of the template DNA
before undertaking primer design. Using in silico molecular biology tools, as discussed below, is a
great way to understand the target sequence you are working with.

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2.1 Best Practices for PCR Primer Design
In order to achieve successful DNA amplification, it’s imperative to understand how to design a primer
and start off with the right primer pair. Here are some primer tips and characteristics you should
consider when designing primers.

Primer Length
The optimal primer length is 18-25 bp. The primer should be short enough to bind easily during
the annealing step, but not too short as short primeres can result in nonspecific binding and thus
inaccurate PCR products. The primer should be long enough for adequate specificity, but not longer
than >30 bp as that may slow down the hybridization rate. Primer length and composition also
directly affects primer melting and annealing temperatures.

Primer Melting Temperature (Tm)

Primer melting temperature is the temperature at which one half of the primer oligonucleotide duplex
(the fragment section where the primer and template have bound) is single-stranded and the other
half is double-stranded, or rather, the primer is about 50% bound to its complementary strand.

Optimal primer pair melting temperatures are between 50° to 60°, and within 5° of each other.
Primer pairs should have similar melting temperatures since annealing during PCR occurs for both
strands simultaneously. Mishybridization may occur if the melting temperature of each primer

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is too high or low relative to the annealing temperature for the reaction. If too low, the formation
of undesired, non-specific duplexes or intramolecular hairpins will occur, or the primers may not
anneal to the template strand at all. If too high, there may be secondary annealing. A primer melting
temperature calculator is found inside of Benchling using your choice of either two algorithms: Saint
Lucia of Modified Breslauer.

Primers with higher G/C content will exhibit higher melting temperatures because G-C hydrogen
bonds are harder to break than A-T hydrogen bonds, thus requiring more energy (a higher
temperature) to melt. If the melting temperature of your primer is very low, try to use a G-C heavy
primer or extend the length of the primer slightly.

Primer Annealing temperature (Ta)

You must take Tm directly into consideration when deciding on annealing temperature. The formula
to calculate annealing temperature is: Ta = 0.3 x Tm(primer) + 0.7 Tm(product) - 14.9

Tm(primer) Tm(product)

Melting temperature of the least stable Melting temperature of the PCR product
primer-template pair

Optimal annealing temperature is discovered empirically, but generally it is less than the primer
melting temperature (Tm) by about 5° - 10°. If primer annealing temperature is too high it’s likely the
primers will not sufficiently bind, resulting in little to zero amplicons. If primer annealing temperature
is too low, primers may begin forming hairpins (discussed more below) or binding to regions outside of
the target sequence, leading to nonspecific, inaccurate PCR products.

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One common way to find the initial optimal annealing temperature is to perform a temperature
gradient PCR, or thermal gradient PCR, starting at about 5° below the lowest melting temperature of
the primer pair.

Primer GC Content and GC Clamp

Primers for PCR and sequencing should have GC content between 40 - 60%, with the 3’ end of the
primer ending C or G to promote binding by DNA polymerase. In the last 5 bases at the 3’ end, there
should be at least 2 G or C bases — this is known as a GC Clamp. G-C bp’s have stronger hydrogen
bonds compared to A-T bp’s (3 hydrogen bonds vs 2), and help with the stability of the primer, and
improve specificity of primer binding.

Set Restriction Enzyme Cut Sites

If using restriction enzyme cut sites in your primer, add 3 to 5 bases to the 5’ end of the cut site. This
is known as the leader sequence and allows for more efficient enzyme cutting. You can easily find
restriction enzyme cut sites via Benchling using criteria such as enzyme names or number of cut sites.

End Stability
Primer end stability is the maximum ΔG value of the five bases from the 3’ end of the primer, or the
maximum ΔG value of where the primer should bind. A stable 3’ end (a more negative ΔG) will help
reduce false priming. Read more about Gibbs Free Energy (ΔG) below.

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2.2 What to Avoid When Designing Primers for PCR
In order to achieve successful DNA amplification, it’s imperative to understand how to design a primer
and start off with the right primer pair. Here are some primer tips and characteristics you should
consider when designing primers.

Repeats/Runs
Avoid runs of four or more of a single base (e.g., ACCCCC), or four or more dinucleotide repeats (e.g.,
ATATATATAT) as they will cause mispriming.

Cross homology
Cross homology is seen when the primer designed is homologous to other regions of the template
strand, and the outcome is amplification of other genes outside the region of interest. Cross homology is
generally avoided by sending the sequence of your designed primers to test its specificity against genetic
databases. By sending primers through the NCBI BLAST software, as you easily can on Benchling, you
can identify regions of significant cross homologies, helping you avoid them in primer design.

Template secondary structure

Similar to primer secondary structures, template secondary structures can be formed during or after
the denaturation phase since ssDNA is generally unstable and can spontaneously fold into secondary
structures. The stability of these template secondary structures depends on their Gibbs free energy
and melting temperatures, similar to the stability of primer secondary structures. If a template
secondary structure forms that is too stable (won’t unfold) even above the annealing temperatures
of the primers, the primers will be unable to bind and PCR amplification will be significantly affected.
Template secondary structure is especially important to consider in qPCR primer design.

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Primer Secondary Structure and Gibbs Free Energy
Secondary structures are created when primers fold in on themselves or bind to each other. Often
called primer dimers, these “bad” primers interfere with primer annealing to the template strand and/
or reduce the availability of “good” primers in the PCR reaction, and ultimately will affect the yield of
high quality PCR products. These are usually caused by intra- or inter-primer homology:

Intra-primer homology Inter-primer homology

A region of 3 or more bases that are Forward and reverse primers that have
complementary to another region within the complementary sequences, causing intermolecular
same primer, causing intramolecular bonding. bonding.

Primer dimer analysis should be performed before using primers in the lab, which includes visualizing
secondary structures and determining the Gibbs free energy (ΔG) for each possible primer dimer. Both
of these primer dimer checks can easily be performed in Benchling. In general, a dimer formed closer
to the 3’ end is much more detrimental than a dimer formed near the 5’ end.

Gibbs free energy ΔG in primer design is the amount of energy needed for a primer to form a particular
secondary structure with itself. In general, ΔG represents the spontaneity of a reaction when held
at a constant temperature and pressure. Structures with a higher ΔG (greater than 0, or positive ΔG)
require an input of energy (heat) to form, so there is a lower likelihood they will form spontaneously
without extra energy. Secondary structures with a lower ΔG (negative ΔG) will happen easily and
spontaneously without additional energy. Very negative ΔG numbers indicate there’s an affinity to
form that structure and it will likely require a lot of heat to reverse the dimer back to linear form, thus
more stable secondary structures (larger negative ΔG values) should be avoided.

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Commonly observed primer dimer secondary structures and their ΔG values in kcal/mol include:

• Hairpins: often formed if there is intra-primer homology within a single primer

and it folds in on itself, or if the melting temperature of the primer is lower
than the annealing temperature of the reaction. 3’ end hairpins are the most
unfavorable as the primer is fully folded back on itself, with the first and last 3 bases being
bound together (not pictured). Generally, 3’ end hairpins with a ΔG of no less than -2kcal/
mol are tolerated. Any more negative and they may not unfold in the PCR reaction. Internal
hairpins (pictured below) are slightly easier to break and a ΔG of no less than -3 kcal/mol is
tolerated during the PCR reaction.
• Self Dimers: formed by inter-primer homology between two identical 5’ 3’
(same sense) primers, when they anneal to each other instead of the 3’ 5’
template strand. In other words, primer self dimers form when a forward
primer anneals to another forward primer, or a reverse primer anneals to another reverse
primer. Self dimers have lower ΔG tolerance than hairpin formations. A self dimer on the 3’
end with a ΔG of no less than -5 kcal/mol, and an internal self dimer with a ΔG of no less than
-6 kcal/mol is generally tolerated.
• Cross Primers: formed by inter-primer homology, but between two different 5’ 3’

primers (a forward primer annealing to are reverse primer). A 3’ end cross 3’ 5’

dimer (pictured) is less tolerated (a ΔG of no less than -5 kcal/mol) than an
internal cross dimer (a ΔG of no less than -6 kcal/mol).

Having a balance of GC-rich and AT-rich domains help avoid secondary structures. Primer design tools
like Benchling can also help detect secondary structures. Do not design primers that allow secondary
structures to remain stable above the annealing temperatures; they will be unable to bind to the
template strand and initiate DNA synthesis, and PCR product yield will be lowered. In other words, if
the ΔG for a secondary structure is so negative (-9 kcal/mol for example), then even the heat of the
annealing temperature will not be able to break the structure back into its original linear form, and it
will not bind to the template strand during the annealing phase.
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Section 3 In Silico Primer Design Tools

In silico modeling and design is a crucial step used both to prepare for and to document a variety of
protocols and experiments done in the lab. Software tools used for modeling range from desktop
applications, to online cloud-based systems, like Benchling. These tools are important for modern
labs as they help maintain institutional knowledge, manage experimental traceability and data, and
automate result capture.

Benchling’s Molecular Biology Suite is a comprehensive set of in silico tools that can model a variety
of protocols beyond primer design, including plasmid design, cloning, sequencing, assembly methods,
CRISPR, DNA alignments, codon optimizations, CRISPR gRNA design, and more. After primer design,
performing an in silico PCR allows scientists to use their amplified gene of interest to model
downstream procedures also, such as cloning or assembly methods.

3.1 Desktop vs. Cloud-Based Primer Design Tools

As R&D workflows increase in complexity in recent years, life science teams have become increasingly
specialized, requiring constant communication with outside partners, other departments, and
upstream or downstream teams. It’s becoming more important to them to find cloud-based tools
that are able to support cross-team collaboration, and that can easily integrate with their broader
informatics infrastructure.

Despite the fact that molecular biology has evolved tremendously over the last decade, most
molecular biology software tools are point solutions that were not built for modern, complex
scientific workflows. With point solutions, R&D organizations may struggle to tie downstream assay
data back to the primers used, or figure out which primers and vectors were already designed and

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stored in their inventory system. Simply, they lack automated traceability of what was performed
after primer design. Many companies are left managing processes using pen and paper, spreadsheets,
and wasting precious lab hours on manual work. This slows down research and introduces risks
into process development and scale up. Desktop point solutions can also be difficult to maintain,
and ultimately do not allow organizations to fully leverage their data and maintain institutional
knowledge long-term.

Primer3 and Primer3Plus

Benchling’s online primer design tool incorporates Primer3
algorithms for automatic primer design to create primers
quickly. Primer3 is a widely used program for designing PCR
primers, hybridization probes, and sequencing primers. In
silico tools provide input to Primer3 of design parameters
determined by the scientist. Primer3Plus, gives a new, more
intuitive web interface to the original Primer3 program.

NCBI Blast
NCBI Blast is a suite of programs started in 1991 by the U.S.
National Center for Biotechnology Information (NCBI). Basic
Local Alignment Search Tool (BLAST) can identify regions of
local similarity between experimental/query sequences and a database of reference sequences. By
comparing nucleotide sequences to different sequence databases, the tool is able to provide detailed
statistical significance reports of any matches. There are various BLAST algorithms, including those
to help identify genomes (RNA or DNA nucleotide sequences), targeted sections like SNPs, and even
proteins. Every query a scientist makes using BLAST is stored forever, creating an ever-growing library
of referenceable sequences that adds to the tools’ accuracy.

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Section 4 Benchling’s Molecular Biology Tools

Primer design may seem straightforward; afterall, primers are less than 30 base pairs long! But
with so many sensitive primer characteristics to consider, along with the sheer volume of primers a
researcher might need to create, it can be daunting to keep the process organized and traceable.

Benchling’s Molecular Biology application enables intelligent primer design, either manually
or automatically with the wizard. With intelligent permission systems, you can set up which
teams have access to which projects. Teams then can save primers in custom or shared
libraries for future reference or further collaboration and design. In the primer tool, Benchling
scans and detects binding sites on sequences, allowing you to accurately attach primers, run
in silico PCR, and use PCR products to plan critical tasks such as digest and ligate methods,
Gibson Cloning, and Golden Gate Assembly.

The application is a powerful tool that’s used for more than just designing primers. With 10+
tools in one unified platform, you can also create sequence annotations, perform alignments,
and design CRISPR guide RNAs (gRNAs). You can easily share and store designs as well, since
the tool is integrated with Benchling’s cloud-based Notebook and Registry.

Construct Design CRISPR Cloning

Restriction-based cloning Guide RNA design Enzyme search

Gibson & Golden Gate assembly On-target / off-target scoring Virtual digests
Bulk assembly HR templates Enzyme cut sites
Codon optimization and Plasmid assembly Ladders library
translation Enzyme lists

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 Sequence Alignment AA / Protein Analysis Sequence Visualization

Alignment to templates AA alignments Plasmid map

Consensus alignments Auto-fill Translations ORF customization
Bulk auto-alignments Antibody Numbering Schemes Annotations
CDR identification Bulk auto-annotations
PTM site identification Sequence search

4.1 Design Primers with Benchling

With Benchling you can easily design and analyze primers
online. Design primers for qPCR, PCR, cloning, and
sequencing. Some highlights include:

• Quickly visualize the primer binding sites on

the sequence of interest and keep track of the
sequences for which the primers are used.
• Easily attach a single primer to a sequence, or
link them in pairs. Primer sequence files, called
“oligos” in Benchling, list all of the sequences to
which they’ve ever been attached, enabling full
traceability for your primer library.

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You can design primers in Benchling either manually or with the Primer Wizard. If your primer is
already designed, you can also easily find any existing primers to attach to your sequence, or import
oligos into Benchling. Let’s see what Benchling’s primer design tool can do.

Figure 6. Primer design in Benchling

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Manual Primer Design
You may want to create primers manually in the Benchling platform from sequences that your
organization already uses. To design primers manually, highlight a region of template DNA on the
sequence map by selecting the desired range, right clicking, and choosing to create either the forward
or reverse primer.

Figure 7a. Manual primer design in Benchling

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Next, in the Design tab, you can focus on the bases of the selected sequence, designate an overhang,
and incorporate restriction enzyme cut sites.

Figure 7b. Manual primer design in Benchling

After designing, quickly verify your primers automatically in the Verify tab. The following are all
accessible within the interface:

• Gibbs Free Energy values for homodimer and monomers

• Melting temperatures and GC content
• Secondary structure diagrams for dimers

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You can even adjust thermodynamic parameters to modify how melting temperature is computed
by clicking the wrench icon. Once design is complete, assign a name to the primer and save it in the
primer library.

Figure 7c. Manual primer design in Benchling

Wizard Primer Design

You can also design primers with the primer wizard, which auto-creates primers for your target
sequence using Primer3 technology. Benchling supports designing three main primer types: PCR,
qPCR, and sequencing primers. Through each step of the wizard, set parameters as needed. Adjust GC
content, melting temperature, length, GC clamp, amplicon length, and more.

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Figure 8a. Primer design in Benchling with wizard

Search, Import, and Attach Existing Primers Easily

If your primer is already designed, with Benchling you can also quickly search, import, and attach any
existing primers.

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Search existing primers that bind to your sequence based on specific primer parameters, such as
length, melting temperature and number of allowed mismatches, and Benchling will show their
stored locations. Select the desired primers and view them bound to the sequence map.

Alternatively, import primers via the Import Oligios feature in Benchling and quickly add any of your
organizations pre-existing primers.

Figure 8b. Primer design in Benchling with wizard

This is especially useful if you are migrating any primers to Benchling from a different tool, such as
Vector NTI, Snapgene, or Geneious. Benchling supports a variety of sequence file types, and carries
over any annotations and tags associated with the sequences.

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Figure 8c. Primer design in Benchling with wizard

Finally, once designed or saved, you can attach primers to your selected sequence. All saved primers
are listed in the primer tool menu for whatever sequence file you are working on.

Attaching existing primers in Benchling allows you to automatically find existing primers that bind to
your sequence, quickly visualize primer binding sites, and view all of the sequence files which any oligo
file (the primer) has been attached to.

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Send Primers to NCBI Blast
With Benchling, a user can easily verify primers’ specificity by sending the oligonucleotides through
the NCBI Blast primer tool. Once inside Benchling, simply click and drag the sequence map to
highlight the primer (or any other sequence of interest), right-click, and send to NCBI Blast. The
sequence is sent to the BLASTN program, which searches against nucleotides.

Figure 9. NCBI Primer Blast Tool in Benchling

NCBI provides the NCBI Primer Blast tool for primer creation, but it requires you to type in or copy/
paste your oligo sequence every time for primer creation. And once created, you must continually
import the designed primer into Benchling or your molecular biology tool. Simply using Benchling will
give you one central location for the entire workflow, while still allowing you to easily send your oligos
to NCBI’s primer blasting tool.

Build Custom Primer Libraries

Share custom primer libraries with your colleagues so you never have to wonder which primers have
already been designed and used in the past. Projects are based on specific permissions, allowing
teams to set up custom libraries as they need. Being able to record and share primers is just one of the
reasons why Eligo Bioscience loves Benchling.

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In silico PCR within Benchling
With Benchling, a user can easily verify primers’ specificity by sending the oligonucleotides through
After designing the primers, you can run PCR in silico with Benchling. Simply select the forward and
reverse primers, link them as a pair and create PCR products with two clicks.

Figure 10. Run PCR in silico within Benchling

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Once functional primers are designed and PCR products (amplicons) are amplified, the Benchling
platform can continue to model downstream procedures such as digestions and ligations, plasmid
design, transfection, and cloning methods such as Gibson and Golden Gate Assembly.

A user can “@” mention any of the PCR products, digests, plasmids, or primer files directly in a
Notebook entry and register them automatically inside the Benchling Registry. Link any of these
in silico procedures to the actual sample used in the lab with the Benchling Inventory. By linking
these designs to physical products in the lab, Benchling gives you a central location to access all
experimental data and share it with different scientists and teams.

Free Primer Design Tools for Academics

Benchling for Academics offers Benchling Molecular Biology – including tools for primer design,
CRISPR gRNA design, alignments, and more – and Benchling Notebook for free to undergraduates,
graduate students, postdocs, and any other academics.

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With more than 180,000 academics using Benchling around the world, we are invested in building the
next generation of biologists. Having access to free, modern, user-friendly tools allows the academic
community to make ground-breaking discoveries faster and spend less time taking notes in paper
notebooks, digging for data across disconnected tools, and completing error-prone manual data entry.
Here are some helpful tips for academics from Benchling for Academics users on how they use the tool
to elevate their research.

Benchling for Academics is also useful to Professors and science teachers. Instructors may now be
faced with the added challenge of teaching courses remotely due to the coronavirus pandemic. Without
access to wet lab courses, it can be tough for students to grasp the concepts behind molecular biology
techniques. But with the right approach, professors and teachers can use Benchling to adapt lab courses
for virtual learning.

Request a Demo of Benchling

If you are interested in learning more about what Benchling can offer your
scientific teams, please request a demo at: benchling.com/request-demo

Primer design is just the beginning of what Benchling offers our customers.
Benchling is a fully integrated life sciences R&D platform that supports
scientist productivity, sample tracking, process management, and analytics.
Used by over 230,000 scientists globally across multinational pharmaceutical
corporations, leading biotech companies, and major research institutions,
Benchling is the leader in cloud informatics for life science R&D.

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Benchling Primer Design and Molecular Biology Resources
• Benchling’s Molecular Biology Tool

• Primers FAQ

• Molecular Biology Overview

• High-Throughput Molecular Biology From Your Living Room

• High-Throughput DNA Sequence Design for Modern Research

• Benchling for Startups: Molecular Biology Deep Drive

• Advanced Molecular Biology Workshop

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