In this exercise we will design oligonucleotide primers to amplify the mature xynB CDS.
The forward and reverse primers will be designed to incorporate attB1 and attB2 sites respectively, to allow clonase-mediated integration of the PCR product into a Gateway entry vector. The primers will be designed to both have a first-round melting temperature (Tm) of 55°C.
The primers will be designed to precisely amplify the mature xynB CDS (no signal sequence) to ensure that when the xynB CDS is transferred into the pDest17 destination vector it will be positioned for in-frame fusion to the vector start codon and associated HIS-tag sequence. This will ensure optimal transcription and expression of the XynB gene product. The xynB CDS stop codon will be included to ensure correct termination of the gene product.
Exercise
1. To start the exercise select the DTU76545 sequence to view it.
2. Click on the yellow xynB mature CDS annotation to select the mature CDS region (positions 139 to 1149).
(If the xynB mature CDS annotation is not visible then click on the icon in the Sequence View side panel and check the option to Show Annotations.)
3. With the xynB mature CDS annotation selected, click the Primers button on the Toolbar and choose Design new primers.
4. In the Design New Primers setup window:
We will design primers with a melting temperature of around 55°C so we need to changes Tm Min: default setttings:
5.To add Gateway extensions to the primers, expand the Advanced section of the Setup window, then click on the 5' Extensions: Fwd: button, click the Gateway site... button and from the dropdown menu choose Site: attB1.
Click OK, and you will see that the extension will comprise 4 G nucleotides and an attB1 site. The G nucleotides are added to ensure the clonase enzyme can efficiently bind to the terminal aatB1 site.
6. For the reverse primer, click the Rev: button, click the Gateway site... button and from the dropdown menu choose to add an "antisense" attB2 site.
Click to run the Primer design tool and two new annotations, 139 F and 1149 R will be added to the sequence. Hit Save (Command or CTRL-S) to save the new annotations.
Hover over each primer sequence and you will see a yellow tooltip containing information about the primer.
7. The next step is to generate a PCR product sequence. Select the DTU76545 file, click on the Primers button and choose Extract PCR Product. The tool will detect the new forward and reverse primer annotations on the sequence and select them. In the dialog that opens, confirm the Forward and Reverse primers are correctly selected, then hit OK.
A new DTU76545 PCR Product sequence will be created. Select it and you should see it has inward facing attB1 and attB2 sites positioned and oriented correctly for a BP clonase reaction.
We will use this PCR product in the next Exercise.
If you plan to routinely design Gateway Primers then you can save your ideal primer design settings by clicking on the cog icon in the bottom left corner of the Design New Primers window and choose to Save current settings. You can load these settings next time by clicking on the cog icon and choosing Load Profile.
To extract your primers to individual files select each primer_bind annotation and Hit the Extract button. You can use these extracted sequences for ordering from your oligo synthesis service provider.
Click the Exercise 2 link below to move to Exercise 2.
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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