TITLE: Overlap extension PCR
AUTHORS: Anna Behle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Linear assembly of PCR fragments.</div><div class = "text-block">Can be used to quickly and efficiently fuse promoters, terminators, fusion proteins etc. without time-consuming sub-cloning steps.</div></div>

[STEPS]
?. [Primer design]
Before starting, thoroughly design and plan your experiment in silico, ideally using cloning software such as SnapGene.Make sure all primers anneal only once in the actual template (e.g. when using a whole genome) and separate parts are able to anneal to each other after PCR. I recommend going through the whole cloning process in silico prior to ordering primers to avoid mistakes. Design primers for all fragments. Primers for overlap extension PCR are designed in a similar manner as for Gibson assembly, except that the outermost parts do not contain overhangs to each other. Annealing parts of primer: Solid arrow in scheme; Should bind to template only once with a Tm of 55-65 ˚COverhang parts of primer: Dotted arrow in scheme; total overlap between two fragments should have a Tm of at least 55 ˚C to avoid unspecific bindingThis method will yield a linear fragment.
It can be beneficial to already add overhangs to the desired vector backbone to the primers flanking the fused part (i.e., P1 and P6 in the scheme) to facilitate downstream cloning.
{"blocks":[{"key":"9jpkc","text":"This protocol summarizes the process of primer design for assembly-based cloning methods, such as Gibson or AQUA-cloning.\u00a0","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"9qqii","text":"Well-designed primers are necessary for efficient cloning of any kind. ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"9civb","text":"This protocol can also be used to design primers for overlap extension PCR of linear fragments. Alternatively, overhangs can also include restriction sites for restriction\/ligation based cloning.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"4i2gl","text":" ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [PCR of parts]
Prepare separate PCR reactions of each part that needs to be assembled.  AB1ComponentAmount [µL]25x Q5 buffer535x High GC buffer54dNTP mix, 10 mM each0.55Primer fwd, 10 µM0.56Primer rev, 10 µM0.57template DNA0.58Q5 Polymerase0.25Gel-purify fragment of correct size.
Since the primers contain long overhangs, it might be necessary to try different annealing temperatures. I generally recommend starting with the annealing temperature corresponding only to the part of the primer which anneals to the template and disregarding the overhang for now.
AB1ComponentAmount [µL]25x Q5 buffer535x High GC buffer54dNTP mix, 10 mM each0.55Primer fwd, 10 µM0.56Primer rev, 10 µM0.57template DNA0.58Q5 Polymerase0.25
?. [Overlap PCR]
Prepare PCR mixture, without primers. Instead of a template, add your PCR parts. Use a large volume, i.e. 1/2 to 3/4 of the total PCR reaction. Make sure to use a molar ratio of ~1:1. Example using Q5-Polymerase:    5x buffer 5 µL   5x high GC buffer 5 µL   dNTPs 0.5 µL   template 4 µL fragment 1 + 5 µL fragment 2 + 5 µL fragment 3   Q5 Polymerase 0.5 µL   H2O to 25 µL    Run your PCR at 15 cycles, using the annealing temperature of the homologous regions.Remove PCR from cycler. Immediately proceed with Step 4.Example using Q5-Polymerase:  AB15x buffer5 µL25x high GC buffer5 µL3dNTPs0.5 µL4template4 µL fragment 1 + 5 µL fragment 2 + 5 µL fragment 35Q5 Polymerase0.5 µL6H2Oto 24 µLRun your PCR at 15 cycles, using the annealing temperature of the homologous regions.Remove PCR from cycler. Immediately proceed with Step 4.
A large volume can be beneficial because often, the concentration of gel-exctracted PCR fragments is low. I recommend using 50 ng of the larger PCR fragment. This step can be varied, but overloading the template DNA can lead to more unspecific product.
AB15x buffer5 µL25x high GC buffer5 µL3dNTPs0.5 µL4template4 µL fragment 1 + 5 µL fragment 2 + 5 µL fragment 35Q5 Polymerase0.5 µL6H2Oto 24 µL
?. [Extension PCR]
Add the two primers flanking the outer parts as you would in a normal PCR.Rerun your PCR at 30 cycles, this time using an annealing temperature matching your flanking primers. Important: Gel-extract your overlap extension product, as this method can result in non-specific side products!PCR reaction will likely yield multiple bands (e.g. the fragments you started out with), as well as a smear around the desired band. Important: Gel-extract your overlap extension product, as this method can result in non-specific side products!PCR reaction will likely yield multiple bands (e.g. the fragments you started out with), as well as a smear around the desired band. This method, when successful, yields a strong band of the correct size that can be used downstream for standard cloning methods such as Gibson Assembly.