Gibson's Assembly
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Gibson Assembly
- 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.
- Digest the vector and do the PCR (don't digest the PCR).
- We usually put 2-3 µg vector in a 50 µL digest.
- 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.
- 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.
- 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).
- Mix together all of the different DNA fragments so that you have equimolar amounts in 5ul total volume (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.
- 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.
- Incubate at 50°C for 60 min (they say you can do 15 to 60 min, but 60 min is optimal).
- 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)