Gateway Cloning Tutorial: Simulating a "LR" Destination clone reaction

In this exercise we will simulate a LR clonase reaction between our new pDONR221 - DTU76545 PCR Product Entry clone and the destination vector pDEST17 vector.

Select the pDEST17 sequence to view it. You will see this vector has attR1 and attR2 sites flanking a chloramphenicol (cmR) resistance gene and a ccdB toxin gene.

To perform the BP clonase reaction, select the pDEST17 and pDONR221 - DTU76545 PCR Product Entry clone sequences then go Cloning → Gateway Cloning. The Gateway cloning tool will identify the att sites present on the entry vector and Destination vector and confirm an LR reaction can be performed. In the test tube an LR reaction creates two new plasmid species. The Gateway tool will output both plasmids if you wish. The tool will ask if you want to "keep both products of the reaction". In this exercise leave this option unchecked so only the final Expression vector will be created.

The tool will confirm sites in each vector are appropriately oriented, and inform that a LR reaction will be performed.

Hit OK and a new 5713 bp plasmid called pDEST17 - pDONR221 - DTU76545 PCR Product Entry clone Expression clone will be created.

The new file name, although descriptive is a bit long and cumbersome. Select the file in the document table, click on the file name to edit it and rename the file to pDEST17:xynB mature.

As a last step we will check that our insert is in-frame with the vector start codon and HIS-tag.

Select the renamed pDEST17:xynB mature sequence, ensure Translation is turned on in the Display tab, then zoom in so that you can see the initiation ATG annotation at the 5' end of the xynB mature CDS annotation. Click on the xynB mature CDS annotation to select it, then drag the left hand boundary of the annotation to extend it to line up with the first "A" of the start codon. You will see in the translation that the start codon and HIS-tag are in-frame with the translated start of the xynB mature CDS "QTSI...".

Note that changing annotations on a sequence with actively linked parents will break the linkage. When you adjust the xynB mature CDS annotation boundary you will get a warning about Actively linked parents. You can ignore this warning and click Continue Editing, just remember to not save the document when asked, or alternately, use menu File → Save As to save an unlinked copy of the original document.


Performing the BP and LR reactions in a single step

To simplify your Gateway reactions it is possible to use the Gateway cloning tool to perform the BP and LR reactions in a single operation. Simply select your PCR product insert with attB sites, your pDONR vector and your pDest vector and go Cloning → Gateway Cloning.

To try cloning in one step, select the DTU76545 PCR Product, the pDONR221 vector and the pDEST17 vector and go Cloning → Gateway Cloning.

The Gateway cloning tool will confirm the selected sequences can be "reacted" and will ouput the same final 5713 bp expression plasmid.

That concludes the exercises on single-insert Gateway cloning. Click the Multisite Overview link below if you want to learn how to do multisite Gateway cloning in Geneious Prime.


General Overview: Overview of tutorial
Exercise 1: Designing primers for Gateway cloning
Exercise 2: Simulating an "BP" Entry clone reaction
Exercise 3: Simulating a "LR" Destination clone reaction
Multisite Overview: Overview of Gateway multisite cloning
Exercise 4: Multisite Part 1: Primer design and PCR
Exercise 5: Multisite Part 2: Creation of Entry and Destination vectors
Gateway Vectors: Preparing Gateway vector sequences

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