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.
- 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)
- amplification of the entire fragment using the outermost primers only.
See this page on Wikipedia for an explanation.
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
- Setup a PCR as normal using Phusion
- Check if the fragments are of the right length on gel
- 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