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.
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.
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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