{line}
{name}
{line}

Plan for the construction of E. coli vector `{rec} <./{rec}.txt>`_

Step 1 PCR of the insert
........................

PCR with primers {fwd} & {rev} and template `{template} <./{template}.txt>`_ results in 
a {length}bp `PCR product <./{pcr_product}.txt>`_


Primers annealing on template:
::

{figure}

Suggested PCR programs for Taq polymerase and for Polymerases with DNA binding domain:
::

{program}

Step 2 Vector digestion and cloning
...................................

Clone the `PCR product <./{pcr_product}.txt>`_ in `pYPKa <./pYPKa.txt>`_ digested 
with `{enz} <http://rebase.neb.com/rebase/enz/{enz}.html>`_ resulting in `{rec} <./{rec}.txt>`_


Step 3 Diagnostic PCR confirmation
..................................

Confirm the structure of the `{rec} <./{rec}.txt>`_ using primers {fp}, {rp} and {f} 
in a multiplex PCR reaction.

Expected PCR products sizes from {fp}, {rp} and {f} (bp):

pYPKa with insert in correct orientation: {correct_products} |br|
pYPKa with insert in reverse orientation: {reversed_products} |br|
Empty pYPKa clone                       : {clone_empty} 


.. |br| raw:: html

   <br />
