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Click OK and the sequence will <strong>be</strong> inserted at the 5' end of the primer.<br />
Perform the same process for the ATP8a1 rev primer, this time using XhoI instead. This time, you<br />
should also add a few bases at the 5' end as was done when inserting the HindIII site to increase<br />
to efficiency of the enzyme.<br />
Note! The ATP8a1 rev primer is designed to match the negative strand, so the restriction site<br />
should <strong>be</strong> added at the 5' end of this sequence as well (using <strong>In</strong>sert Restriction Site <strong>be</strong>fore Selection).<br />
Save the two primers and close the views.<br />
2.3 Simulate PCR to create the fragment<br />
Now, we want to extract the PCR product from the template ATP8a1 mRNA sequence using the<br />
two primers with restriction sites:<br />
Toolbox | Primers and Pro<strong>be</strong>s | Find Binding Sites and Create Fragments