Gateway Cloning Tutorial: Simulating an "BP" Entry clone reaction

In this exercise we will simulate a BP clonase reaction between our new PCR product and a donor vector with appropriate attP1 and attP2 sites.

Select the pDONR221 sequence to view it. You will see this vector has attP1 and attP2 sites flanking a chloramphenicol (cmR) resistance gene and a ccdB toxin gene. This region will be replaced by recombination with the PCR product to create an entry vector.

To perform the BP clonase reaction, select the pDONR221 and DTU76545 PCR Product sequences then go Cloning → Gateway Cloning. The Gateway cloning tool will identify the att sites present on both the vector and the PCR product insert, confirm they are appropriately oriented, and inform that a BP reaction will be performed.

Hit OK and a new sequence called pDONR221 - DTU76545 PCR Product Entry clone will be created. This new 3558 bp sequence represents the expected entry plasmid generated by the clonase reaction. You will see that the clonase reaction has created flanking attL1 and attL2 sites suitable for use in a clonase-mediated LR-reaction.

The pDONR vectors manual recommends that individual E. coli colonies containing your clonase-derived constructs are checked by PCR using the M13-F (-20) and M13-R primers. Binding sites for these primers are present and annotated onto the pDONR221 vector. We will now use the Extract PCR Product tool to simulate this PCR reaction so that we can determine the size and sequence of the predicted product.

Select the new pDONR221 - DTU76545 PCR Product Entry clone, Hit the Primers button and choose Extract PCR product. Set the Forward primer to M13-F (-20) and the Reverse primer to M13-R (-26). This will create a 1303 bp PCR product sequence.


Click the Exercise 3 link below to move to Exercise 3.


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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