Pre-requisites and 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.
- How to manually design primers
- Generate a PCR fragment in silico
- Integrate primers, fragments and protocols into lab notebooks
Before you begin:
This learning module contains links to files on Benchling that are uneditable. You will need to sign up for a Benchling account to create your own copy of these files that you can edit. Sequences can be copied directly into your Projects while notebook entries will require manual copy & paste into a new blank entry.
Design primers and simulate PCR products in silico
PCR is a common molecular biology technique for amplifying genetic material. 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. For this exercise we will use this PCR template.
- Click+drag your mouse over a region to highlight bases to obtain primer properties (such as Tm, length, GC content)
- Once highlighted, click the "Create" button -> "Primer" → "Forward"
- This will open the Design Primer tab where you can name and save your primer sequence.
- The forward primer you’ve generated for this PCR template. Repeat these steps except use "Reverse" when creating the 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 onto 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.
Extension exercise/ Stretch yourself
Take it a step further by showing students how to integrate this with the Notebook experimentally especially when carrying out PCR reactions.
- 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.
- Encourage students to type a brief summary of what criteria they used to create their primers and PCR product.
Use the Benchling Notebook to plan and record PCR experiments
You can use the Notebook stand-alone to record experimental protocols and data. If you don’t feel like writing out experimental protocols for PCR, you can attach protocols from the literature or from a manufacturer and view them inside of your entry. This is useful so you don’t need to spend time rewriting you protocols especially if you’re using it from a specific source.
When setting up experimental PCRs, teach your students about PCR mastermixes. A “mastermix” combines common reagents 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.
Congrats! You've finished the learning module: Primer Design and PCR.
Learn about our next molecular biology technique on Benchling: Gel Electrophoresis and Restriction Enzymes