Editing Gibson Assembly (section)

== Gibson Assembly Protocol for Joining Two or More PCR Fragments (Gene Synthsis) ==

1) Prepare 6 ml of '''5X ISO Buffer''' in a 15 ml falcon tube as follows:

3 ml '''1 M Tris-HCl pH 7.5'''

+ 150µl 2 M MgCl<sub>2</sub>

+ 240 µl 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)

+ 300µl 1 M DTT

+ 1.5 g PEG-8000

+ 300µl 100 mM '''NAD'''

+ dH<sub>2</sub>0 to 6 ml

Store at -20 <sup>o</sup>C in 320µl aliquots.

2) Prepare 0.5 ml of '''Gibson assembly master mix '''as follows:

320µl 5X ISO Buffer

+ 0.64 µl 10 U/µl T5 exonuclease*

+ 20 µl 2 U/µl Phusion polymerase

+ 160 µl 40 U/µl Taq ligase

<strike>+ dH<sub>2</sub>0 to 1.2 ml</strike>

Store at -20 <sup>o</sup>C in <strike>15</strike> 5 µl aliquots.

This is optimized for 20-150 bp sequence homology overlaps

3) Thaw a <strike>15</strike> 5 µl aliquot of the Gibson assembly master mix, and keep on ice until use.

4) Add purified PCR fragments, or unpurified PCR products

5) Add water to <strike>20</strike> 16 µl

6) Incubate at 50 oC water bath for 1 hour.

7). Run agarose gel and cut out right size band, use Qiagen agarose gel extraction kit to purify DNA

8). Digest with enzyme and clone into a digested vector.





<strike>5) Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to the thawed 15µl master mix in a 20 µl total volume assembly reaction mixture as follows:</strike>

<strike>linearized vector backbone (100 ng)</strike>

<strike>+ each additional assembly piece (to equimolar with backbone)</strike>

<strike>+ 15µl Gibson assembly master mix</strike>

<strike>+ dH<sub>2</sub>0 to 20µl</strike>

<strike>6) Incubate the assembly reaction at 50 oC for 15 ~ 60 minutes, and then place on ice.</strike>

<strike>7) Transform 5 ~20 µl of the assembly reaction into 100µl of competent E. coli and/or run an agarose gel to check for successful assembly.</strike>

<strike>A major limitation (especially in high GC rich templates) to Gibson assembly is that the termini of the DNA sequence fragments to be assembled should not have stable single stranded DNA secondary structure, such as a hairpin or a stem loop, or repeated sequences, as this would directly compete with the required single-stranded annealing/priming of neighboring assembly fragments.</strike>