Virtual Cloning Tutorial - Introduce Cut Site using PCR

We now need to check that the PCR product that our primers will produce is in the correct frame to produce a functional GFP-fusion protein once ligated into the vector.

Select the reverse primer, which contains the NcoI site at the 5' end, turn on Complement in the sequence view options and note how many bases are introduced between the primer binding site and the cut site on the reverse strand (1 base).


Thus, the process of PCR and restriction digest with NcoI will introduce two extra bases to our human proinsulin gene, one at each end. This will cause a frameshift and destroy our protein because it is not a multiple of 3.

To rectify this, turn on Allow Editing and insert another G (C on the complement) just between the NcoI recognition sequence and the primer binding site. Click Save and our primers are ready to be tested.


Select Human Proinsulin again and delete the two primer annotations.

Test the new primers by going to Primer→Test with Saved Primers. Choose your forward and reverse, turn off the Included Region, Target Region and Product Size options if they are on, and click OK.


Hover your mouse over the reverse primer annotation and in the pop-up containing the primer statistics note the "Extension" which we added which contains the NcoI restriction site.


Now use Primers→Extract PCR Product to pull out the PCR product. You don't need to select forward and reverse primers because there are only two. Note that the result will now have NcoI sites on both ends.



Exercise 5: Insert into Vector