Gibson Assembly

Background on Gibson Assembly

https://www.youtube.com/watch?v=f984D89MqfI

https://www.youtube.com/watch?v=tlVbf5fXhp4

https://www.biocat.com/bc/files/Gibson_Guide_V2_101417_web_version_8.5_x_11_FINAL.pdf


Gibson Assembly

1. Design your insert(s) so that there is ~25 bp of homology at each point of recombination.
   • It is fine to have more homology than this, the protocol says that you can use 20 to150 bp with reaction mixture that we are using.
   • If you are having a hard time getting it to work (or doing more fragments at once) it is probably worth it to go to 40-50 bp of homology
     • Experience has shown 25 bp to be good enough even for multi-piece assembly. We don’t generally worry about the Tm of the homologous regions, but I would imagine you could have problems if it’s extremely AT-rich. Presumably the Tm just needs to be > 58 °C.
2. Digest the vector and do the PCR (don't digest the PCR).
   • We usually put 2-3 µg vector in a 50 µL digest.
3. PCR purify the vector and PCR products as you normally would (eluting in 30ul).
   • Gel purification results in lower background but also fewer colonies. If you are getting a lot of background you can gel purify stuff, but since it is annoying to do so and it often works with PCR purification I would try that first.
4. Quantify the vector digests and PCR's.
   • It doesn't appear necessary to get great quantitation, so you can do it on the Nanodrop, but I have found that I get somewhat better results when I quantify it for real (on a gel).
     • Most people in our lab just combine 4 µL of PCR-purified vector with 1 µL of gel-purified insert, for two piece cloning, without even bothering to check concentrations. I have seen molar ratio affect the # of colonies you get, but have never had a ratio change make the number of colonies go from zero to more than zero, with a given vector/PCR prep.
     • If you do want to calculate an equimolar concentration for the fragments, I find it convenient to use units of “ng/kb” – just divide through your mass concentrations from Nanodrop by the size of that fragment.
5. Mix together all of the different DNA fragments so that you have 5:1 ratio of moles of insert to moles of vector (3:1 for inserts larger >300 bp). A calculator for small inserts can be seen below. Total volume should be 5ul (in a PCR reaction tube).
   • It’s not worth doing a negative control. You tend to get plenty of colonies on the no-insert plate, but your hit rate on the with-insert plate will be much higher than the control plate would suggest; not sure why.
6. Add one aliquot (15ul) of Gibson one-step isothermal reaction mix.
   • The aliquot contains a lot of PEG, so be sure to mix the aliquot well before pipetting it out of the tube and after adding it to the DNA.
   • The protocol works because the exonuclease does not work well at 50°C, so when preparing the reaction I always keep the tube on ice and avoid working with it at room temp.
     • Or don’t mix until you’re ready to put the tube immediately into a 50 degree heat block / thermocycler / whatever.
7. Incubate at 50°C for 60 min (they say you can do 15 to 60 min, but 60 min is optimal).
8. Transform
   • Do the full 20ul reaction in chemically competent cells

This is the one-step isothermal DNA assembly protocol from: Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343 - 345 (2009) (http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html)

Gibson Assembly -- Making Aliquots

1. Mix:
   1. 320ul of 5x Isothermal Reaction Buffer (see below)
      • This is very viscous, vortex well before pipetting
      • Each aliquot is used once; throw it away after use (there will still be ~160ul left)
      • Do not use sonication when thawing the 5x Isothermal Reaction Buffer
   2. 700ul sterile water
2. Vortex and spin down in centrifuge
3. Add:
   • 0.64ul T5 Exonuclease
   • 20ul Phusion Polymerase
   • 160ul Taq Ligase (not T4 ligase; this must be Taq ligase)
4. Mix by pipette and spin down
5. Aliquot 15.6ul aliquots into sterilized 600ul microcentrifuge tubes (~66 reactions)
6. Freeze aliquots at -20 degrees

Gibson Assembly -- 5x Isothermal Reaction Buffer

1. Mix the following filter-sterilized solutions:
   • 3mL of 1M Tris-HCl pH 7.5 (at room temperature)
     • Add 1.5g PEG-8000 and dissolve by incubating in the 55 degree water bath
     • Allow to cool to room temperature before adding the rest of the reagents
   • 150ul 2M MgCl2
   • 60ul 100mM dATP
   • 60ul 100mM dCTP
   • 60ul 100mM dGTP
   • 60ul 100mM dTTP
   • 300ul 1M DTT
   • 300ul 100mM NAD+
   • Fill to 6mL (unknown with what... probably water?)
2. Make sure everything is in solution
3. Do not cool to 4 degrees, as the PEG will precipitate. If you can spin it down, that is ideal, but if not, then just vortex it and then get as much solution as possible to the bottom
4. Aliquot into 12 x 450ul aliquots and store at -20 degrees

Gibson Assembly Volume Calculator

http://165.82.245.159:5000/d/s/501837446508492808/mfeJSwDlpF0Cqn3GCVJiv6_IKj7rALz5--7AA.Rzj9gY_