PCR and Primer Design

Before you begin

Learn how to get started and sign up with Benchling here. Start this worksheet using your free academic account in order to get the most out of this worksheet. DNA sequences can be copied directly into your Benchling account. For notebook entries, you can manually copy & paste the content into a new blank entry.

Content and materials for this module were co-developed with Dr. Philip Leftwich, Biology Lecturer at the University of East Anglia (Norwich, UK)

Overview

This worksheet teaches you how to design primers on Benchling for applications such as PCR. This can be combined with writing out PCR protocols and data collection in your electronic lab notebook to gain confidence in experimental design and generate a reproducible lab project.

Prior knowledge

A basic understanding of the concept of Polymerase Chain Reaction – and familiarity with common terms such as DNA template, primers – for background on the theory of PCR see this video.

Learning outcomes

• Manually design primers

• Generate a PCR fragment in silico

• Integrate primers, fragments, and protocols into lab notebooks

Worked Example

Design primers and simulate PCR products in silico

PCR is a common molecular biology technique for amplifying DNA. PCR products and the primers necessary to generate them can be designed and modeled in silico on Benchling. This tutorial will guide you through the process of manually designing primers on a DNA template for PCR.

In this PCR template, you will see that there are multiple pre-designed primers attached to the template sequence. Find the annotation for pBAD_fwd_primer and pBAD_rev_primer. Note the following properties for each:

pBAD_fwd_primer:
LENGTH: 20
GC: 35%
MELTING TEMP: 48°C

pBAD_rev_primer:
LENGTH: 18
GC: 33.33%
MELTING TEMP: 40.8°C

What makes a good primer?

Some guidelines for designing PCR primers include:

1. Length of 18-24 bases (Specificity usually is dependent on the length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.)

2. 40-60% G/C content (Be mindful not to have too many repeating G or C bases, as this can promote primer-dimer formation.)

3. Start and end with 1-2 G/C pairs (This is known as a GC Clamp).

4. Melting temperature (Tm) of 50-60°C (This can depend on your PCR buffer conditions and DNA polymerase as well).

5. Primer pairs should have a Tm within 5°C of each other

6. Primer pairs should not have complementary regions

Question(s)

How many of these guidelines does the pBAD fwd & rev primer pair meet?

Try to answer this question on your own and check the "Solution" at the bottom.

Practice

Can you design a primer pair that amplifies a similar region of the DNA template which meets more of these basic criteria above?

For designing primers that bind to your target sequence, you can create primers manually through the following tutorial starting with the PCR template from earlier.

• Click+drag your mouse over a region to highlight bases to obtain primer properties (such as melting temperature (Tm), length, GC content)

• Once highlighted, click the "Create" button -> "Primer" → "Forward" (Alternatively, you can right-click on the sequence and follow the same prompts)

• This will open the Design Primer tab where you can name and save your primer sequence.

• You have now generated the forward primer needed for PCR.

• Repeat these steps except select "Reverse" when creating the reverse primer for this sequence.

Once attached, you can link your primers and simulate the expected PCR product on Benchling. Once your primers have been linked, this will create a “Primer Pair” and Benchling will calculate the expected basepair length of your product.

• Look to the right sidebar and click on the "Primer" icon. Hover over your forward primer and left-click it.

• Now hold "Shift" down and left-click on the reverse primer

• Switch to the "Pairs" tab and click on "Link Primers" to pair these primers together. Then scroll down and select "Create PCR Product".

• Ignore the items in the next menu and just select "Copy" and save this new sequence. You can rename the file later to reflect that it’s your expected PCR product.

Stretch yourself

Take it a step further by integrating these DNA sequences with the Benchling Notebook. This is especially useful when combined with experimental lab notes that accompany your PCR reactions.

• Open a new lab notebook entry

• PCR products and designed primers can be referenced in notebook entries by dragging and dropping a sequence inside or use @mentions to “@” and type the name of the sequence.

• Type out a brief summary of what criteria you used to create your primers and include information such as the expected size of your PCR product.

• Voila! Now you have notes that are connected to your primer designs.

Use the Benchling Notebook to plan and record PCR experiments

When setting up experimental PCRs in the lab you will need to assemble a PCR master mix. A “mastermix” combines common reagents (dNTPs, buffer, water, and DNA polymerase) that are aliquoted out to ensure consistent reaction efficiencies and reduce overall mixing steps. You can use tables on Benchling to input formulas and automate volume calculations for the reagents in your PCR mastermix. Check out the example notebook entry and see for yourself; PCR Protocols and Mastermix Calculator.

You can use the Notebook stand-alone to record experimental protocols and data. Save time from rewriting out published protocols by inserting them into your notebook. You can attach protocols from the literature or from a manufacturer and view them inside your entry. This is useful as you don’t need to spend time writing & rewriting your protocols, especially if you’re using them from a specific source.

Solution(s)

The primer pair pBAD fwd and rev and guidelines they follow:

1. Yes; both have a length between 18-24 bp.

2. No; both have GC content below 40%.

3. Yes, both end on a GC clamp.

4. No, both have a Tm of below 50°C.

5. No, both primers have greater than a difference of 5 °C in their Tms

6. No, they do not have complementary regions.

Note: This doesn’t mean this primer pair won’t work - but the more guidelines you can stick to when designing primers, the more likely your amplification will be successful.

Congrats! You've finished the learning module: Primer Design and PCR.