Editing Isothermal Assembly (section)

== Protocol  ==

'''Short'''<br> 

1. PCR vector and insert(s) ensuring that at least 40 bp homology exists between adjacent fragments<br>2. Thaw assembly master mix and keep on ice until ready to be used<br> 3. Mix 15 ul of assembly mixture with 5 ul total of cleaned PCR product (PCR Cleanup Kit or Gel Extraction) keeping DNA inserts in equimolar amounts<br> 4. Incubate at 50 °C for 15-60 min (60 min optimal).<br> 5. Transform cells with no more than 1 ul of assembly mixture. 

'''Long'''<br> 

1. PCR up your fragments of choice, and gel purify. 

2. Not exceeding a total volume of 5 ul, in a PCR tube, combine fragments at equal molecular ratio [e.g. amount fragment1 = 100 ng * (fragment 1 size / backbone size); 100 ng * (fragment 2 size / backbone size); backbone = 100 ng]. If required, bring to 5 ul with ddH2O. I recommend using approx. 100 ng of plasmid backbone (fragment containing antibiotic resistance). Quantitative fusion of two linear PCR fragments is also possible (incubation &gt;30 min will lead to degradation). 

3. Add the combined fragments (5 ul) to 1 Isothermal Assembly reaction aliquot (15 ul) and mix by pipetting (20 ul total). 

4. Place rxn. at 50C for 15 -30 min. 

5. (optional for chem. transform.) Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 ul of ddH2O. 

6. Transform with 1 ul of assembly rxn. <br>