Week 1: Design primers for PCR

1. Import pUC-GFP into Benchling

a. Click the " button on the left-hand panel, "Sequence," "Import from File/DB"

b. Search "https://www.addgene.org/11155/" and save it into your project folder

Annotations from the external database will be automatically imported

2. Find the region you want to amplify

a. Click on the "Annotations" tab on the right-side panel (first button)

b. Click on "EGFP" to highlight

3. Use the Primer Wizard to find appropriate primers for the EGFP region

a. Click on the "Primers" tab on the right-side panel (third button), "Create Primers," "Wizard"

b. Make sure that the "Task" is set to "PCR," and click "Use Selection" to specify that the target region is EGFP

c. Modify the parameters as necessary (if you don't change any settings, the wizard will automatically set the optimal Tm as 62 C, GC content as 50%, and size as 22bp)

d. Click "Generate Primers," select which primer pair you'd like to use, and click "Save Selected Primers"

4. Order your primers

a. Saved primers are automatically attached to your pUC-GFP sequence. To access your saved primers, click on the "Primers" tab

b. Click "Export Primers" to easily copy and paste information into an order form

5. Visualize your PCR product on Benchling

a. Click on the "Primers" tab and click "Pairs"

b. Select the primer pair, and click "Create PCR Product"

Week 2: Perform PCR and verify products

6. To perform three PCR reactions, create a Master Mix of PCR reactants for three, and aliquot the Master Mix to three tubes of 50µL each

7. Perform PCR using these thermal cycling conditions

8. Verify successful PCR amplification via gel electrophoresis

9. Optional: Purify PCR product, and sequence the result

a. Compare sequencing result to expected PCR product using Benchling's alignment tool