Stitch PCR

In order to avoid having to do cloning we sometimes use an 'Overlap Extension PCR' or 'stich PCR'. This means we use two consequetive PCRs.

1. independent amplifications of each of the fragments that need to be combined using primers with tails that overlap with the fragment from the other PCR(s)
2. amplification of the entire fragment using the outermost primers only.

See this page on Wikipedia for an explanation.

Primer design

Design primers for each of the fragements as you always would. Use a Tm of around 60C.

These primers can also be used for ligations using the Gibson protocol.

---> stch1_Fw                                           ---> stch2_Fw
===========================================             ====================================
                              stch1_Rv <---                                   stch2_Rv  <--- 



---> stch1_Fw                                       -------> stch2_Fw
===========================================             ====================================
                              stch1_Rv <-------                               stch2_Rv  <--- 

• Primer stch1_Fw and stch2_Rv stay the same
• Take the reverse complement of stch1_Rv and add it at to the 5` of stch2_Fw
• Take the reverse complement of this newly created long primer which then becomes stch1_Rv

PCR 1

• Setup a PCR as normal using Phusion
• Check if the fragments are of the right length on gel

PCR 2

• Setup another Phusion PCR, like normal, but using a 1:10 dilution of the products of PCR 1 as template.
• In this PCR we will use only the outer primers (stch1_Fw and stch2_Rv)
• Check if the fragment is of the right length on gel
• Sometimes this PCR results in multiple band (especially when combining more than two fragments). Possible solutions in order of likely:
  • work with a lower dilution of the PCR1 fragement
  • Do a cleanup the product from PCR1 to remove any leftover primers
  • reduce the number of cycles of PCR2