Smolke:Protocols/DNA assembly

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(Gibson isothermal in vitro DNA assembly)
(Gibson isothermal in vitro DNA assembly)
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The 1x concentration of the mix is 10 ul. So to carry out an assembly, add a total volume of 2.5 ul of prepped, linear DNA to the mix and incubate at 50C for 30-60 minutes and then directly transform the mix into heat-shock competent ''E. coli'' and plate onto the appropriate selective solid medium.
The 1x concentration of the mix is 10 ul. So to carry out an assembly, add a total volume of 2.5 ul of prepped, linear DNA to the mix and incubate at 50C for 30-60 minutes and then directly transform the mix into heat-shock competent ''E. coli'' and plate onto the appropriate selective solid medium.
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'''Tips'''
'''Tips'''
*If you get too few colonies or no colonies - try adding more DNA. Either concentrate DNA samples before adding or just add more DNA to the mix (you can go a bit, 2-3ul, above the 10ul without ruining the Gibson reaction)
*If you get too few colonies or no colonies - try adding more DNA. Either concentrate DNA samples before adding or just add more DNA to the mix (you can go a bit, 2-3ul, above the 10ul without ruining the Gibson reaction)
*If you are getting many negative colonies, specifically uncut backbone plasmid, in your transformations, try increasing the ratio of insert fragment DNA to backbone plasmid DNA
*If you are getting many negative colonies, specifically uncut backbone plasmid, in your transformations, try increasing the ratio of insert fragment DNA to backbone plasmid DNA
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 +
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'''Making the mix'''
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''Adapted from Gibson Nature Protocols April 2009''
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*320ul 5X ISO buffer (made identically as listed in the paper)
 +
*0.64 ul T5 Exonuclease
 +
*20 ul Phusion (special phusion - ask Mike about it)
 +
*40 ul Taq ligase (less than in paper - see note)
 +
*820 ul water
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This is 1.2 ml of Gibson mix - the tricky part is aliquoting out the 160 individual PCR tubes with 7.5 ul each. Find an aliquoting buddy!
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*Gene assembly protocol page updated and moved here:
*Gene assembly protocol page updated and moved here:
[[Smolke:Protocols/Gene assembly|Gene assembly]]
[[Smolke:Protocols/Gene assembly|Gene assembly]]
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*We use 1/4 the Taq ligase prescribed in the paper - for cloning into TOP10, the assembly mix works without any Taq ligase. I think this is because the cells can accept nicked plasmid DNA. We have been using this lower amount of Taq for the last ~2 years, so seems to work well and reduces the cost per reaction by ~60% -Mike

Revision as of 20:33, 17 February 2014

Gibson isothermal in vitro DNA assembly

We now maintain a stock of "Gibson mix" for multi-fragment DNA assembly as an alternative to traditional cloning for the Smolke Lab.

The mix is stored in 7.5 ul aliquots in PCR tubes in the bottom of the metabolic engineering -20C freezer.

The 1x concentration of the mix is 10 ul. So to carry out an assembly, add a total volume of 2.5 ul of prepped, linear DNA to the mix and incubate at 50C for 30-60 minutes and then directly transform the mix into heat-shock competent E. coli and plate onto the appropriate selective solid medium.


Tips

  • If you get too few colonies or no colonies - try adding more DNA. Either concentrate DNA samples before adding or just add more DNA to the mix (you can go a bit, 2-3ul, above the 10ul without ruining the Gibson reaction)
  • If you are getting many negative colonies, specifically uncut backbone plasmid, in your transformations, try increasing the ratio of insert fragment DNA to backbone plasmid DNA


Making the mix Adapted from Gibson Nature Protocols April 2009

  • 320ul 5X ISO buffer (made identically as listed in the paper)
  • 0.64 ul T5 Exonuclease
  • 20 ul Phusion (special phusion - ask Mike about it)
  • 40 ul Taq ligase (less than in paper - see note)
  • 820 ul water

This is 1.2 ml of Gibson mix - the tricky part is aliquoting out the 160 individual PCR tubes with 7.5 ul each. Find an aliquoting buddy!


Notes

  • As of 2-17-2014 Taq ligase is already included in the Gibson mix aliquots, so no additional enzymes need be added
  • Gene assembly protocol page updated and moved here:

Gene assembly

  • We use 1/4 the Taq ligase prescribed in the paper - for cloning into TOP10, the assembly mix works without any Taq ligase. I think this is because the cells can accept nicked plasmid DNA. We have been using this lower amount of Taq for the last ~2 years, so seems to work well and reduces the cost per reaction by ~60% -Mike
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