Multisite Gateway cloning Exercise Part 2: Creation of Entry vectors and destination vector

Part 2: Creation of Entry vectors

We will now combine our new PCR products with the appropriate donor vectors using the Gateway Cloning tool.

Select the new trpA1 UP PCR product and the pDONR221 P1-P4 vector and go Cloning → Gateway Cloning. This will create a new circular vector called pDONR221 P1-P4 - trpA1 UP PCR Product Entry clone.

Select the new GAL4 PCR product and the pDONR221 P4r-P3r vector and go Cloning → Gateway Cloning tool. This will create a new circular vector called pDONR221 P4r-P3r - GAL4 PCR Product Entry clone.

Select the new trpA1 DOWN PCR product and the pDONR221 P3-P2 vector and go Cloning → Gateway Cloning tool. This will create a new circular vector called pDONR221 P4r-P3r - GAL4 PCR Product Entry clone.

We now have 3 pDONR entry vectors that can be recombined with an appropriate destination vector in a multisite reaction.

Part 3: Creation of the final destination vector

To create the final destination vector, select pDESTHaw vector and the three new entry clone sequences. Go Cloning → Gateway Cloning. If all vectors have compatible attB sites then you will see the following dialog:

Click OK and a new 15,894 bp circular sequence will be created called pDESTHaw - Reaction of 3 fragments Expression clone.

Rename the sequence to something more useful, select the pDESTHaw - Reaction of 3 fragments Expression clone.

Performing the multisite BP and LR reactions in a single step

To simplify your Gateway multisite reactions it is possible to use the Gateway cloning tool to perform all BP and LR reactions in a single operation. Simply select your three PCR products generated earlier with attB sites, the corresponding 3 pDONR vectors and the pDestHaw desitination vector and go Cloning → Gateway Cloning.

The Gateway cloning tool will confirm the selected sequences can be "reacted" and create the same 15,894 bp pDESTHaw-based expression plasmid.

That concludes the exercises on Multisite Gateway cloning.


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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