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    PCR amplification of GFP
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    PCR amplification of GFP

    Introduction

    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 To edit this protocol, sign up/sign in with Benchling, click the clock icon on the top right, and click the Clone From Version button.

    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
              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
              1. 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
              1. 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 22 bp)
              • d. Click "Generate Primers," select which primer pair you'd like to use, and click "Save Selected Primers"
              1. 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
              1. 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
              1. To perform three PCR reactions, create a Master Mix of PCR reactants for three, and aliquot the Master Mix to three tubes of 50 uL each
              Table3
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              A
              B
              1
              		Master mix (# of rxns)3
              1 row
              Table1
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              A
              B
              C
              D
              1
              		Reagentsfinal conc.1x (uL)master mix
              2
              		water37.5112.5
              3
              		10X buffer1x515
              4
              		dNTP mix (10 mM)200 uM13
              5
              		forward primer0.1~1 uM2.57.5
              6
              		reverse primer0.1~1 uM2.57.5
              7
              		DNA template0.5ug/50uL13
              8
              		Pfu polymerase (2-3u/uL)1.25u/50uL0.51.5
              8 rows
              1. Perform PCR using these thermal cycling conditions
              Table2
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              A
              B
              C
              D
              E
              1
              		#StepTemperatureTime#of Cycles
              2
              		1Denaturation95 C2 min1
              3
              		2Denaturation95 C1 min25
              4
              		3Annealing55 C30 sec25
              5
              		4Extension72 C3 min25
              6
              		5Final extension72 C5 min1
              7
              		6Hold4 Cindefinite1
              7 rows
              1. Verify successful PCR amplification via gel electrophoresis
              1. Optional: Purify PCR product, and sequence the result
              • a. Compare sequencing result to expected PCR product using Benchling's alignment tool

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