Virtual Cloning Tutorial - Cloning Primer Design

Open Human Proinsulin if it isn't already open.

Turn on Translation with By selection or annotation in the sequence viewer options.

Select the region from the beginning of the NcoI binding site to just before the stop codon of the CDS (54 to 385). Don't worry that the translation in the selected region is wrong since it is the location of the stop we're interested in.


Select Primers→Design New Primers

Design forward and reverse primers with Included region 54-385, Target region off, Product size 332 in both boxes and Number of pairs 1. Click More Options and drop Tm Min to 45.


By turning off the target region and forcing the product size to be the length of the selected region this forces the primers to be designed at the ends of the selected region as required for cloning primers.

Click OK, then save the document.

Save the two primers by selecting each in the sequence view, clicking Extract and giving them meaningful names. The reverse one will ask if you want to reverse complement it. Answer yes otherwise the sequence view won't agree with the text view. Delete the CDS annotations from the primers to save confusion at a later stage.


Exercise 5: Introduce Cut Site using PCR
Exercise 6: Insert into Vector