Editing Isothermal Assembly (section)

== <br>Comments  ==

*When preparing the isothermal reaction mix, add&nbsp; PEG slowly to liquid. If added too quickly it will form a plug which will make mixing difficult<br> 
*The original paper suggests that 10 - 100 ng of total DNA be used for assemblies. However as high as 170 ng can be added<br> 
*This protocol can be&nbsp; used for a two way and three way ligation.<br> 
*There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. It was reported that of 50% of assemblies could a missense mutation. Therefore it is advised you sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors.<br> 
*Both purified PCR product (using PCR cleanup kit) and gel purified DNA fragment could be used for assembly. PCR cleanup gives more colonies (more DNA, better quality, i.e., no agarose/QG contamination), but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used.<br> 
*Primers with 20 bp homology and a 20 bp spacer from the adjacent fragment can be used for assembly. <br>

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[[Category:Protocols]]