Restriction cloning (R10+)

This tutorial shows you how to use the restriction cloning tool in Geneious R10 and later.

DOWNLOAD THE RESTRICTION CLONING TUTORIAL

Tutorial Instructions

Geneious Prime tutorials are installed by either 'Dragging and dropping' the zip file into Geneious Prime or using File → Import → From File... in the Geneious Prime menu. Do not unzip the tutorial.

Restriction Cloning Tutorial

Note: To complete the tutorial with the referenced data please download and install the tutorial above into Geneious Prime.

The Geneious Prime Restriction Cloning tool can ligate any combination of linear or circular nucleotide sequences, will automatically identify compatible cut sites, and perform one-step digestion and ligation with drag and drop ordering of two or more fragments. 

Input documents do not need to be predigested or annotated with restriction sites, as Geneious will identify the restriction sites present in your input documents and attempt to guess the sites your cloning strategy will use.  By default, restriction sites with recognition sites of 4 bp or less are not considered.  If you need the Restriction Cloning tool to use 4 bp cutters then contact the Geneious support team.

The Restriction Cloning tool can be found under menu Tools → Cloning, or via the Cloning button on the Toolbar. You can launch the Restriction Cloning tool with or without selecting your input sequences first.

In this tutorial we will ligate a PCR product into the multiple cloning site (MCS) of the expression vector pET26B (See Novagen). The pET26B vector MCS lies within a short coding sequence (CDS) that encodes an N-terminal PelB signal sequence for potential periplasmic localization and a C-terminal His•Tag® sequence.

The PCR product provided in this tutorial has been generated with PCR primers designed to amplify the region encoding the mature XynA enzyme (without signal peptide or stop codon) from the bacterium Dictyoglomus thermophilum.  The primers were designed with extensions to generate an N-terminal NcoI site and a C-terminal BamHI site.  The NcoI site has been positioned so that ligation will give an in-frame fusion to the pET26B-based PelB signal peptide.  The BamHI site is positioned so that ligation will generate an in-frame C-terminal fusion of XynA to the pET26B-based HIS-tag.

Exercise 1: Creating restriction enzyme subsets
Exercise 2: Performing Restriction Cloning
Exercise 3: Confirming CDS in-frame fusion
Troubleshooting

Exercise 1: Creating a restriction enzyme subset for cloning operations

To increase the chances that the restriction cloning tool will successfully identify the sites you want to use, it is best to create restriction enzyme subsets for your commonly used cloning vectors.  You only need to create these lists the first time you perform a restriction cloning operation.

To create a Restriction enzyme subset do the following.

1.  Select the pET26B document provided with this tutorial (selecting any sequence will also work).

2.  Go Cloning → Find Restriction sites.  This will pop out the Restriction Cloning tab associated with the sequence viewer panel.

3.  Set Candidate enzymes: to commercially available enzymes.

4.  Click on the Advanced button to bring up the list of all enzymes in the commercially available enzymes set.

The pET26B multiple cloning site (MCS) provides the following 11 restriction sites for cloning operations:  BamHI, EagI, EcoRI, HindIIIMscI, NcoI, NdeI, NotI, SalI, SacI (SstI) and XhoI.  We will now create a restriction enzyme subset that comprises only these sites.

5.   Click the check box at the top of the list to deselect all enzymes in the list, then find and select the 11 enzymes for our subset.  Click Save Selected Enzymes and name the list pET26B MCS RE sites.

Click OK, the Done. This will create a new Enzyme Set file that can be used for all future restriction cloning operations using the pET26B vector.  All files of this type are indexed so they can be used from anywhere in your Geneious database regardless of the folder they are located in. 

Exercise 2: Performing Restriction Cloning

A: Overview of The Restriction Cloning window

The Restriction Cloning window comprises 4 main sections.

The top section  allows you to set your vector (Backbone), define how the Candidate Enzymes are chosen, and if chosen from an Enzyme Set, define which enzyme set to use.

The panel below this, referred to as the “Construct layout” panel  shows tag representations of all sequences selected for the cloning operation.  You can use the Choose… or Add Insert…buttons to add a new backbone or new insert sequences.

You can drag and drop tags to change their order, and click on the tag-associated triangle to see a dropdown menu that allows you to switch sites, reverse complement the sequence or modify digestion overhangs on each tag.

Any tag selected in panel  will be displayed graphically in the “Detailed view” panel .  The region of the sequence that will be involved in the cloning operation will be highlighted by a thick blue line.

The bottom section  provides options for how the Restriction cloning operations results are output.

You can click on the Help button in the bottom left corner of the window for more information on all of these options

B: Performing Restriction Cloning

The following steps describe how to perform restriction cloning using the example sequence documents provided with this tutorial.

1.  Select the pET26B vector and xynA PCR Product files that are provided with this tutorial.

2.  Go to Cloning → Restriction Cloning to open the Restriction Cloning setup window.  pET26B should be selected as the backbone and shaded blue in the Construct Layout panel, and you should see “Use Leftmost” in the Backbone dropdown menu. You can also choose a specific document from this menu if it is not set correctly.

3.  Set the Enzyme set to the pET26B MCS RE sites list.  The Restriction Cloning tool should then correctly identify and select the NcoI and BamHI sites intended for this cloning operation. You can also see and select other restriction sites by clicking the down arrow on each document tab in the layout window

4.  If you are satisfied the settings will correctly simulate your cloning operation, then go OK.  A new file called pET26B – xynA_PCR_Product will be created.

Exercise 3: Confirming the CDS in-frame fusion

As mentioned in the introduction to this tutorial, the input PCR product for this demonstration was designed to generate an N-terminal and C-Terminal in-frame fusion between the mature xynA CDS and the CDS that spans the MCS of pET26B.

To check that the in-frame fusions have occurred correctly:

1.  Select the pET26B-XynA_PCR_Product document created by the cloning operation. The concatenation annotations (purple) show you where the PCR product was inserted into the vector.

2.  Locate and select the CDS (yellow) annotation called MCS CDS PelB-MCS-His-tag.  If required, click on the Annotation tab and select it from the Annotation list.  This should select two intervals generated due to insertion of xynA. . Option (ALT)-click on the annotation to zoom in on this region.  

3.  Go menu Edit -> Go to Base, then click Go to select the CDS intervals and the intervening sequence (5,072 to 6,175). Click on the Add Annotation button, and add a new annotation called xynA fusion, of type CDS and go OK.  Note that you will get a warning that the document has an Actively linked parent. Continue with the editing.

4. A new CDS will appear.  Hover over the new CDS to see the automatic translation and confirm the CDS translates correctly over the entire CDS range. Save the document, and choose to save a copy.

5. Select and delete the preexisting CDS’s and delete them so only the XynA fusion CDS remains.  Turn on Translation in the General tab, select the Annotations tab and turn on display of Ligation annotations, then zoom in to view the translation at the fusion sites and confirm the gene product is correctly translated through the ligation sites.

Troubleshooting

This section of the tutorial describes how to correct things when Geneious Prime incorrectly selects the wrong restriction enzymes, or selects the wrong restriction digestion fragment for your ligation.

1.  Select the pET26B vector and xynA PCR Product files again.

2.  Go Cloning → Restriction Cloning to open the Restriction Cloning setup window.

3.  Click on Enzyme Set and set the Enzyme set to the commonly used enzymes list.

The pET26B vector also has a single BglII site.  BglII and BamHI have compatible cohesive ends.  If you examine the regions and sites selected by Geneious for the cloning operation, you will see that it has incorrectly guessed the BglII site should be used for the cloning operation, resulting in a short (176 bp) BglII-NcoI fragment being selected for ligation.

4.  To correct the site selection, right-click on the triangle on the pET26B tag and select Choose enzyme cut site for the 5′ reaction. 

5.  Choose BamHI from the list of available sites and go OK.  This should restore the correct sites and allow you to proceed with the restriction cloning operation.