Editing Isothermal Assembly

== Gibson Chew Back And Anneal Assembly: One Step Isothermal Assembly  ==

Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes. In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment per interface needs to have an overlap extension into the adjacent fragment). These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase. In theory, many fragments can be assembled simultaneously (5, 4 inserts + backbone, have been documented in the initial report) into a single product of up to ~100 kb+. The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.<br> 

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Isothermal Assembly works by combining a cocktail of exonuclease, polymerase, and ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT overlap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50C. 

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== Reagents  ==

'''5x Isothermal Reaction Mix''' 

3 ml 1 M Tris-HCl (pH 7.5), autoclaved<br> 300 μL 1 M MgCl<sub>2 </sub>(autoclaved)<br> 60 μL 100 mM dGTP<br> 60 μL 100 mM dATP<br> 60 μL 100 mM dTTP<br> 60 μL 100 mM dCTP<br> 300 μL 1 M DTT<br> 1.5 g PEG-8000<br> 300 μL 100 mM NAD (MW: 663, 250 mg to 3.77 ml H<sub>2</sub>O)<br> Add autoclaved ddH<sub>2</sub>O to 6 ml in total 

<br>'''Assembly Master Mix''' 

320 μL 5X Isothermal Master Mix <br> 0.64 μL 10 U/μL T5 exonuclease <br> 20 μL 2 U/μL Phusion DNA Pol <br>160 μl 40 U/μl Taq ligase<br>

700 μL ddH2O to 1.2 ml Total 

Aliquoted reaction as 15 ul aliquots (80 tubes)

'''For 0.75 ml Master mix'''<br>

240 μL 5X Isothermal Master Mix<br>0.5 μL 10 U/μL T5 exonuclease<br>15 μL 2 U/μL Phusion DNA Pol<br>100 μl 40 U/μl Taq ligase

395 μL ddH2O to 750 μL Total <br>

Aliquoted reaction as 15 ul aliquots (50 tubes). <br>

Master mixes are stable at -20°C and can withstand several freeze thaw cycles.<br><br> 

== 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> 

== <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]]