Exercises for the Cloning tutorial

Go to parent CLC Bio Main Workbench

Loading sequences in CLC

Some of the sequences that are going to be used in the exercises are in the /Documents/CLC/ folder. They were generated in CLC and exported as .clc files to allow for easy and complete import during the training. As an example, we will import the sequence of primer ATP8a1_fusion_fwd.

Import the primer sequence.

Click the Import button in the top toolbar Browse to the .clc file containing the primer (on the BITS laptops you can find it in C:\Users\bits\Documents\CLC\ATP8a1fusion_fwd.clc), select the file and click Next. Indicate the folder in the Navigation area where you want to store the new primer, in our case that's the Primers folder in the Cloning folder in the Example Data. Click Finish. The primer now appears in the chosen folder in the Navigation area. You can double click it to open in the View area.

Loading vector sequences from other resources

CLC comes with a list of built-in vector sequences: expand Cloning vector library in the Cloning folder in the Example Data.

However, the list of built-in vector is very limited, most vector sequences have to be imported from Addgene, an organization that improves sharing of plasmids.

In CLC you can load sequences directly from Genbank or you can load them by importing a file. Even if you don't load them from Genbank it is still a good idea to import them in Genbank format (and not in Fasta format). Genbank format not only contains a sequence but also all annotations. If you load the annotations (in Genbank format) CLC will automatically show them.

Obtain the annotated sequence of pAcGFP1-C1 from Addgenes.
On the webpage of pAcGFP-C1 click Sequence next to Analyze: On the next page select the Sequence tab: To include the annotation, you need the sequence in Genbank format: Copy the text and paste it in Notepad. Save the file as pAGFP1C1.txt

Open the annotated sequence of pAcGFP1-C1 in CLC.

Click the Import button in the top toolbar Browse to the pAcGFP1C1.txt file, select the file and click Next. Indicate the folder in the Navigation area where you want to store the new primer, in our case that's the Cloning folder in the Example Data. Click Finish. The vector now appears in the chosen folder in the Navigation area. You can double click it to open in the View area. Switch to the circular view of the vector by clicking the Show Circular View button in the bottom toolbar.

Loading a full list of annotated vector sequences

Download a list of annotated vector sequences from Invitrogen. Make sure you save it as a .ma4 file.

Load the list in CLC.

Create a new folder by right clicking CLC-Data in the Navigation Area. Select New and Folder. Provide a name for the new folder and click OK. Click the Import button in the top toolbar. Browse to the .ma4 file, select the file and click Next. Indicate the folder in the Navigation area where you want to store the vector list, in our case that's the newly created folder. Click Finish. The vectors now appear in the chosen folder in the Navigation area.

Changing annotation of sequences in CLC

Although we imported pAcGFP1-C1 in Genbank format, the annotation from the file was not completely correctly imported. What is called ORF frame 1, ORF frame 2 and ORF frame 3 in the map is redundant and should be removed.

Remove the ORF frame annotations.
Right click ORF frame 1 in the map and select Delete Annotation: Do the same for the other 'ORF frame' annotations and save the vector.

Searching vectors containing a certain sequence in CLC

You can search vectors containing a certain sequence, e.g. all vectors that contain a TATA box, via a motif list. This can be helpful to automatically annotate the TATA box in all vectors, also in the vectors where it hasn’t been annotated yet.

Motifs are sequence patterns: they can be represented by a string (a word) or by a regular expression. Regular expressions are flexible words e.g. %car% is a regular expression that represent all words that contain the word car like cart, acaricide…).

Create a motif list
In the top menu select Toolbox, then General sequence analyses and Create Motif List. This will open an empty list.

The regular expression for the TATA box is TATA[AT]A[AT]A (Java regular expression format).

Add the TATA box motif to the motif list
In the top menu select Toolbox, then General sequence analyses and Create Motif List. Select Java regular expression Provide a name for the motif e.g. TATA box Provide the regular expression of the motif

• Simple motifs consist of text, e.g. ATGATGNNATG (you can use N’s to add some flexibility)
• More information on regular expressions in Java format.
• More info on regular expressions in Prosite format.

Now you can search for the motif in this motif list using the motif search in the Toolbox. This will search for motifs and displays the results in an overview table. This is particularly useful when searching for motifs on many sequences.

Search for the TATA box motif in the list of vector sequences from Invitrogen

In the top menu select Toolbox, then General sequence analyses and Motif Search. Use the arrows to add all sequences of the Invitrogen vector folder and click Next. Select to search for a MotifList. Click the small search button to select the saved motif list. Accuracy applies only to simple motifs and not to regular expressions so we don't need to set this parameter. Search for reverse motif enables searching on the negative strand on nucleotide sequences. Select this. Exclude unknown regions only applies to motifs containing Ns so we don't need to set this parameter. Click Next. Select to Create a table containing the results of the search. Click Finish.

If you are doing this to annotate TATA-boxes in the vector sequences select to Add annotations instead of Create a table. This will add an annotation to the sequence when the motif is found.

Primer design in CLC

The Workbench is well equiped for designing primers for many applications. However, when specificity of the primers is crucial, e.g. for qPCR, it is much easier to use Primer Blast.

Go to the Primer design in CLC tutorial for an overview of how to design primers in CLC.

In CLC you can define many criteria that the primers have to satisfy, allowing you to design very efficient pairs of primers. However, CLC does not allow you to define criteria for the specificity of the primers as Primer-BLAST does. CLC does not guarantee that the primers are specifically targeting the region you want to amplify. If you want to design primers for amplifying a region from the genome (or the transcriptome) you have to check the specificity of the primers to avoid decreasing the efficiency of the primers and generating aspecific PCR products.

Restriction cloning in CLC

Cloning is easier in SnapGene than in the Workbench so we generally recommend to do the cloning in SnapGene. To convince you of this we will do a restriction cloning with a single fragment in CLC, very similar to the exercise in SnapGene.

Restriction cloning of a single fragment

Go to the Restriction cloning of a single fragment in CLC tutorial for an overview of how to insert a fragment into a vector using restriction enzymes.

Restriction cloning of multiple fragments

Go to the Restriction cloning of a multiple fragments in CLC tutorial for an overview of how to insert a fusion of two fragments into a vector using restriction enzymes.

Fusion-based cloning in CLC

In-Fusion cloning, (cloning of fragments into a linearized vector based on PCR using pimers that overlap with the ends of the vector) is as such not supported in CLC. You can do it but you have to perform each step manually.

We will list the steps so you can do it if needed. However, since it is supported in SnapGene and it is relatively easy to do in SnapGene, we recommend using SnapGene for this.

Go to the Fusion-based cloning in CLC tutorial for an overview of how to insert a fragment into a vector using PCR.

Gateway cloning in CLC

The Workbench does support in silico Gateway cloning. Gateway cloning in the lab is done as follows:

• attB sites are added to a sequence fragment
• the resulting attB-flanked fragment is recombined in an entry vector (BP reaction) to construct an entry clone
• the target fragment from the entry clone is recombined into a destination vector (LR reaction) to construct an expression clone

As an example we will clone the CDS of Atp8a1. In the example we will clone a single fragment, but the Gateway system allows cloning of multiple fragments.

Add attB sites to the insert

The first step is to amplify the target sequence with primers containing attB sites.

Obtain the CDS of Atp8a1.
Open the Atp8a1 mRNA sequence. Right click the CDS and select Open Annotation in New View. Save the sequence as Atp8a1 CDS.

Add attB sites to the Atp8a1 CDS.

Open the Gateway cloning editor by expanding Toolbox in the top menu, select Cloning and Restriction Sites (red), then Gateway Cloning and finally Add attB Sites (green) This opens a window where you can select one or more sequences. If you are cloning multiple fragments, you should select them all at this point. Select Atp8a1 CDS and click Next. This opens a window where you can choose which attB sites you wish to add to each end of the fragment. If you have several fragments and wish to add different combinations of sites, you will have to run this tool once for each combination. Click Next to add sequences to the primers between the template specific part and the attB site. For example add a Kozak sequence between the attB and the gene of interest. Press Shift – F1 and you will see a list of possible additions from which you can choose. This list shows you the most common additions, but you can manually type or paste whatever sequence that you want to add. At the bottom of the window, you can see a preview of what the final PCR product looks like. In the middle is the sequence of interest (Atp8a1 CDS). In the beginning is the attB1 site and at the end the attB2 site. The primer additions you have inserted are shown in color. Note that the four terminal G’s are automatically added to the attB sites. Click Next to specify the length of the template-specific part of the primers. The Workbench is not doing any primer design. You specify the length of the part of the primers that should base pair with the Atp8a1 CDS, together with the attB sites and additions, and these are the primers. The Workbench will not check the compatibility of the primers, the annealing temperatures or the GC-content.Click Next. Choose to Open the results in the Workbench. Click Finish. The main output is the input sequence now including attB sites and primer additions. There will be one output sequence for each sequence you have selected for adding attB sites. You also get a list of the primers.

Save the resulting sequence as it will be the input to the next part of the Gateway cloning workflow.

Perform the BP reaction

The next step in the Gateway cloning workflow is to recombine the attB-flanked fragment into an donor vector to create an entry clone, the so-called BP reaction. First, you have to download the entry vector (pDONR221) from Addgenes or Invitrogen and import it into the Workbench since it’s not yet present in the Cloning vector library in the Navigation Area.

To save time, the vector was already downloaded and is present in the /Documents/CLC folder. Import the sequence into the Workbench. Make sure that you circularize the vector and save it before you proceed with the actual cloning.

Perform the BP reaction.

Open the Gateway cloning editor by expanding Toolbox in the top menu, select Cloning and Restriction Sites (red), then Gateway Cloning and finally Create Entry Clone (BP) (green) Now you can select the fragment to be recombined into the donor vector. Note that the fragment should be flanked with attB sites. If you want to clone multiple fragments, you can select more than one sequence as input. For each input fragment an entry clone will be created. Click Next. Select Atp8a1 CDS (attB1 attB2) and click Next. Now you have to specify the donor vector that you are going to use. Click the browse button to select a donor vector. Click Next, choose to Open the results and click Finish. The Workbench looks for the attP sites in the sequence of the donor vector so you should make sure that attP sites are present in the donor vector you choose. It only checks that valid attP sites are found - it does not check that they correspond to the attB sites of the selected fragments at this step. If the right combination of attB and attP sites is not found, no entry clones will be produced.

The output is one entry clone per fragment (in our case one entry clone). The attB and attP sites have been used for the recombination, and the entry clone is now equipped with attL sites. The by-product of the BP recombination is not part of the output. Save the results since you will need them for the subsequent LR reaction.

Perform LR reaction

The final step in the Gateway cloning workflow is to recombine the entry clone into a destination vector to create an expression clone, the so-called LR reaction.

I would have liked to do the exercise on the same destination vector as in SnapGene, pEarleyGate 103, but the vector was not available for import in the Workbench. The only place (apart from the SnapGene website) where I could find them is the website of the creators but this is a raw text version without annotation.

So we will use a standard Gataway destination vector, pDEST14, available at Addgene. To save time, the file is already on your computer in the /Documents/CLC/ folder. Import the vector into the Workbench. Follow exactly the same procedure as for the entry vector. Don’t forget to circularize the vector and save it.

Perform the LR reaction.

Open the Gateway cloning editor by expanding Toolbox in the top menu, select Cloning and Restriction Sites (red), then Gateway Cloning and finally Create Expression Clone (LR) (green) Now you can select the entry clone to be recombined into the donor vector. If you want to clone multiple fragments, you should select multiple entry clones here. When you have selected the entry clone, click Next. Now you can select a destination vector. Click Next, choose to Open the results and click Finish. The Workbench looks for the attR sites in the sequence of the destination vector so you should make sure that attR sites are present in the destination vector you choose. It only checks that valid attR sites are found - it does not check that they correspond to the attL sites of the selected entry clone at this step. If the right combination of attR and attL sites is not found, no expression clones will be produced.

The output is an expression clone. The attL and attR sites have been used for the recombination, and the expression clone is now equipped with attB sites.

When performing multi-site Gateway cloning, the Workbench will insert the fragments (contained in separate entry clones) by matching the sites that are compatible. If the sites have been defined correctly, an expression clone containing all the fragments will be created.