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Gibson Assembly Master Mix

Gibson Assembly Master Mix

11 Comments
  1. julio permalink

    Hi Ethan,
    Thanks for your protocol. Just wanted to make sure here if it is 3ml of 1M Tris-HCl, pH8.0? Because it seems to be quite concentrated in the 2X master mix too.

  2. ethanomics permalink

    Hi Julio,
    Thanks for spotting the omission. Yes, that should be 1 M Tris-HCl, pH 8.0. The final 1X concentration is 100 mM which does seem high but it works. I’ve updated the protocol. I should have referenced this in the post but it came from here:
    http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html
    But I didn’t like how they used dNTP’s, which fall into the category of expensive reagents, in their 5X Isothermal Reaction Buffer and then only a small fraction of that was used in the final master mix. I also wanted a 2X mix like what NEB sells and not a 1.33X mix.

    Ethan

    • julio permalink

      Thanks Ethan,
      Also I think you are missing out on the “Add water to 6 ml” in your ISO mix. Maybe I am wrong, but something does not add up. Thanks Again.

  3. ethanomics permalink

    Hi Julio,
    There is no water to be added to ISO mix but you are right, something doesn’t add up and that is the MgCl2. The recipe should say 150 ul of 2 M MgCl2 and NOT 1 M MgCl2. My apologies for the mistake (I am the undisputed king of the typo) and thanks for pointing that out!!! I’m pretty sure that I made up my 2X master mix with 1 M MgCl2 and it did work better then NEB’s master mix with two different assemblies so it may be an improvement. Regardless, next time make it up I will definitely use 150 ul of 2 M MgCl2.

    Another good thing about blogging, free proofreading of your protocols.

    Ethan

  4. julio permalink

    Hi Ethan!!
    I was playing around with this… this is not too surprising but worth mentioning….
    I used NEB’s Q5 polymerase and it’s own Q5 buffer supply with of PEG8000, T5 exo, Taq ligase and NAD+ in to a 2X master mix and it worked as well as the gibson kit sold!!!! It is super cheap and easy to make it!!!

    happy cloning,
    julio

  5. ethanomics permalink

    It has come to my attention that there is an additional mistake in my 2X Gibson Assembly Master Mix assembly (Thanks Steve!!!). My previous recipe had 10X more T5 exonuclease then what was published. I don’t know what was going through my head when I wrote this one up. Usually I just have a bunch of dyslexic typos scattered all over the place but not stuff that matters. Well the good news is the lab has been using these homemade mixes and they work better then the stuff NEB sells. But I think in the future we will be using the formulation as is described in the Gibson paper. I’ve updated the recipe, but I haven’t used the updated version yet. Since this one is actually what was published I don’t imagine any problems with it.

  6. Wong permalink

    Hi julio,

    Can you share more details about how you did the 2x master mix using Q5 buffer? I bought the Q5 polymerase with the buffer, but I cannot find the composition of the buffer from their product description. Do you mean in the buffer the PEG 8000 is already included? What about the others: Tris-HCl, MgCl2?? Would you please share how you exactly made your master mix. Thanks

    • julio permalink

      Hi Wong,
      for a 40ul RXN, I use 1U T5 exo, 160U Taq ligase, 0.5U Q5 Pol ( remember not to use the hotstart version), supply with 5% PEG and 1mM NAD+ at final concentration. It works very well in my hands – julio

  7. Julio – Can you explain further your protocol? For 40 uL reaction you seem to be using a lot more T5 exo and Taq ligase than the normal protocol, is this correct?

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