PCR amplification of GFP
Introduction
Materials
- pUB-GFP plasmid
- PFU DNA Polymerase 10X buffer with MgSO4
- dNTP mix (10mM each)
- PFU DNA polymerase
- Water
Procedure
Week 1: Design primers for PCR
- 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
- 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
- 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"
- 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
- 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
- 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
A A | B B | |
1 1 | Master mix (# of rxns) | 3 |
A A | B B | C C | D D | |
1 1 | Reagents | final conc. | 1x (uL) | master mix |
2 2 | water | 37.5 | 112.5 | |
3 3 | 10X buffer | 1x | 5 | 15 |
4 4 | dNTP mix (10 mM) | 200 uM | 1 | 3 |
5 5 | forward primer | 0.1~1 uM | 2.5 | 7.5 |
6 6 | reverse primer | 0.1~1 uM | 2.5 | 7.5 |
7 7 | DNA template | 0.5ug/50uL | 1 | 3 |
8 8 | Pfu polymerase (2-3u/uL) | 1.25u/50uL | 0.5 | 1.5 |
- Perform PCR using these thermal cycling conditions
A A | B B | C C | D D | E E | |
1 1 | # | Step | Temperature | Time | #of Cycles |
2 2 | 1 | Denaturation | 95 C | 2 min | 1 |
3 3 | 2 | Denaturation | 95 C | 1 min | 25 |
4 4 | 3 | Annealing | 55 C | 30 sec | 25 |
5 5 | 4 | Extension | 72 C | 3 min | 25 |
6 6 | 5 | Final extension | 72 C | 5 min | 1 |
7 7 | 6 | Hold | 4 C | indefinite | 1 |
- Verify successful PCR amplification via gel electrophoresis
- Optional: Purify PCR product, and sequence the result
a. Compare sequencing result to expected PCR product using Benchling's alignment tool
Comments
Welcome to this example protocol from Benchling for Education— easily share protocols like these with your entire class by signing up for Benchling!
Purpose: To amplify GFP from the pUB-GFP plasmid via the polymerase chain reaction (PCR)
Skills learned in this module:
- Design primers necessary to amplify pflp-1
- Perform PCR
- Verify PCR products via gel electrophoresis
You can order the pUB-GFP plasmid from Addgene here
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