Editing Ligation and enzyme-independent assembly methods (section)

== Overlap extension cloning ==

<br/>1. Perform PCR as above.

<br/>2. Analyse products by agarose gel electrophoresis as above.<br/>If non-specific products or primer-dimers are present it is essential to remove them by PCR optimisation, gel extraction as above, or T4 treatment.

<br/>3. Column purify megaprimer and quantify by spectroscopy.&nbsp;<br/>For products <500 bp, 1 µL of PCR product can be used directly for thermal cycling&nbsp;without purification. Reduced efficiencies may be observed for larger fragments.

<br/>4. Perform overlap extension linear amplification.<br/>The optimal megaprimer concentration to use is: <350 bp, ~100-300 fmol (5-15 nM);&nbsp;<br/>&lt;1.5 kb ~50-100 fmol (2.5-5 nM); &gt;1.5 kb, ~25-50 fmol (1.25-2.5 nM).<br/>To remove primer-dimers with T4 DNA polymerase, treat a 10 µL fraction of&nbsp;1x SmartCut buffer containing ten-fold the optimum megaprimer in 10 µL, incubate<br/>with 3 U (1 µL) of T4, incubate for 30 min at 25 °C and immediately use 5 µL for&nbsp;overlap extension.

<br/>Perform thermal cycling according to manufacturer’s instructions with a high fidelity, non-strand-displacing DNA polymerase, such as Phusion Hot Start II High Fidelity<br/>DNA Polymerase, a 20 µL reaction volume, 25 ng of plasmid template and 5%&nbsp;DMSO.

<br/>An example thermal cycling program for Phusion:<br/>72 °C 5 min (to blunt the megaprimer),<br/>98 °C 3 min,<br/>(98 °C 30 s 63 °C 30 s 72 °C 45 s/kb) x 30

5. Dilute 5 uL of product with 5 uL CutSmart Buffer (to maximise DpnI activity and reduce inhibition by PCR buffer) and 20 U (1 µL) DpnI and incubated for 1-3 hr at<br/>37 °C.

<br/>6. Transform bacteria as above.