Gibson Assembly & Primer Design in Your Browser

Transcript

1. 0:00

   Introduction: Gibson Assembly & primer design

   In this video I show how to perform a Gibson Assembly and design the primers for it. First we need a plasmid from Addgene.

2. 0:06

   Import a plasmid directly from Addgene (#52691)

   We take Addgene plasmid 52691, pREP42-MCS+, and import it. You can import it directly, or first add your Addgene username and password in Settings.

3. 0:27

   Connect your Addgene account in Settings

   I have added my Addgene username and password in Settings, so the plasmid imports straight from Addgene. Now say we want to place this nmt1 promoter in front of a gene in a genome region from NCBI, so I import a second sequence.

4. 0:55

   Import a genome region from NCBI (S. pombe ade6)

   I choose Genome region. Under Locus, in the reference genomes, the species is S. pombe, so I search for pombe and pick it. Then the gene: ade6.

5. 1:21

   Set up PCR and open the primer designer

   Now I want to design primers for this. I click here, choose PCR, and then Design primers.

6. 1:41

   Choose Gibson Assembly (or PCR / homologous recombination)

   There are several kinds of primer design. Since we are doing a Gibson Assembly, we choose Gibson Assembly; you can also design for a normal PCR or for homologous recombination, and the steps are mostly the same. We give the inputs, one and two, in the order they should be joined, so that once the primers are designed they are filled in automatically. I click Design primers and it switches to the primer design section.

7. 2:19

   Select the nmt1 promoter region

   First choose what to amplify: the nmt1 promoter. To be realistic, we design the primers just around the promoter. Click the feature, switch to the sequence, and the nmt1 region is already selected. You can also click to place the cursor and amplify the whole plasmid from there. The start and end positions are filled in.

8. 3:08

   Choose the insertion site and check orientation

   Next, choose where it goes: the ade6 gene. I click it and it is selected, and the preview shows the product, with the promoter just before the gene. The nmt1 promoter is already in the right orientation here; you can reverse it and watch it flip.

9. 3:44

   Generate and save the primers

   I click Design primers, and these are the primers. Once we save them, they are added to the PCRs automatically.

10. 3:52

    Run the PCRs with automatic annealing temperatures

    The primers arrive with their annealing temperature, so I run a PCR on each input.

11. 4:00

    Run the Gibson Assembly and view the product (circular/linear)

    Then the Gibson Assembly: the two PCR products are already selected, so I submit. The product is ready, and you can view it in linear and circular view.

12. 4:17

    Save the construct and add it to your lab notebook

    I rename it to Gibson and save it, and it opens as a tab. Zoomed in, there is a button that adds it to a notebook: I add it to the Gibson Assembly entry and save. Open the entry, scroll down, and the product is there in the notebook.

13. 4:58

    Share the cloning workspace with colleagues

    Back in the workspace where I ran the reactions, I can share it with colleagues: create a link, add their email, and choose whether they can view or edit.

14. 5:16

    Open the result in the sequence editor

    The product can be viewed in more detail too: zoom in, click it, and it opens in the sequence editor.