I am using the NEB HiFi DNA Assembly Master Mix to assemble 4 fragments (about 1000bp for each) to pUC19(2700bp). The total length is about 7500bp. But I tried several times, I didn't get any colonies. My ratio is 1:1:1:1:1. I have checked my overlaps and the length of overlap is 35-65bp and Tm is about 70 degree and GC content is 40-60%. Can anyone give me some advice about my questions. Thank you.
Hi Liya,
one thing that might be helpful is to divide your gibson into two reactions, combining them upon 15min. Try to keep GC as low as possible and load app. +-50ng material (equimolar). You can always load your gibson assembly on bioanalyzer to figure out which part is problematic.
I hope this helps, M.
Hi Liya,
in our lab there are three frequent causes when Gibson assembly does not work:
- the vector is not linearised completely (50 ng of the linearised vector usually works fine)
- if you purify your fragments for assembly after the initial PCR-amplification via agarose gel, make sure that you yield enough DNA. If you measure concentration (photometer, nanodrop) make sure that there is really a peak for nucleic acid (260 nm). Sometimes after gel elution DNA concentration is overestimated a lot due to contaminants. I often check the DNA amount via agarose gel.
- sometimes you are unlucky and one of the primers adding the overhangs to the next fragment was synthesized incorrectly. Even if you use HPLC purified primers, they are only check for length not correct sequence. Once I had such a problem which suddenly was solved by ordering the same primer sequence again.
Good luck!
No colonies doesn't mean the assembly failed. It means your transformation procedure isn't good enough to see it.
If your transformation was up to snuff, you would be seeing false assemblies and vector background colonies (even when, and especially when the assembly fails!). If the assembly works, then you get additional colonies containing the correct product on top of that. It's virtually impossible to get zero colonies with a commercial, well-characterized cell that's certified for large plasmids.
Your correct product might well be there. If you can fix your transformation, you can find out.
John is of course completely right, checking competent cells/transformation procedure is very easy (use a plasmid roughly the same size as the one you want to assemble). In our lab we always use self-made chemical competent cells and never face any problems even using large plasmids.
Hi Liya,
You might have a problem with your transformation. I have had toxicity problems with the Gibson Assembly kit and certain types of competent cells (such as Stabl3 cells). You can use the NEB equivalent of Stabl3 cells (https://www.neb.com/products/c3040-neb-stable-competent-e-coli-high-efficiency)
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