Editing Ligation and enzyme-independent assembly methods (section)

== PIPE cloning without purification ==

<br/>1. Amplify the insert and vector by PCR using a minimum amount of vector plasmid&nbsp;template (0.5 ng template/50 µL reaction) according to manufacturer’s instructions&nbsp;with a high fidelity DNA polymerase, such as Phusion Hot Start II High Fidelity&nbsp; DNA Polymerase, but omit the final extension step. An enhancer such as 5% DMSO&nbsp;can be added to increase robustness.<br/>An example thermal cycling program for Phusion:<br/>98 °C 3 min,<br/>(98 °C 30 s 63 °C 30 s 72 °C 45 s/kb) x 35

2. Check the products on an agarose gel for successful amplification. If vector&nbsp;plasmid amplification failed, cut the vector with a restriction enzyme within the&nbsp;multiple-cloning site and repeat the PCR using more template.&nbsp;If non-specific products are observed, you may wish to optimise the PCR conditions&nbsp;or gel extract the correct fragment for maximum cloning efficiency. For gel extraction&nbsp;of the correct fragment, avoid exposure to damaging short-wave UV light, such as by&nbsp;using methylene blue staining with visible light, or loading duplicate samples, cutting&nbsp;the gel in half between them and marking the transilluminated half as a reference.

<br/>3. Combine 5 µL of vector and 5 µL of insert PCR product (or vary accordingly for&nbsp;varying yields by band intensity to get close to a 2:1 molar ratio) with 10 U (0.5 µL)&nbsp;of DpnI and incubate at 37 °C for 1-3 hr. To save time, digestion can be performed&nbsp;during gel electrophoresis.

<br/>4. Transform ultracompetent bacteria with the digest mixture up to 10% of the final volume of cells according to manufacturer’s instructions. For example, add 2 µL&nbsp;DNA to 18 µL of XL10 Gold cells&nbsp;