Good day to all. I am encountering a problem in determining the correct amount of plasmid and inserts' concentration. According to the Gibson assembly protocol, I should include a 0.2-1 pmol of vector for an assembly of 4-6 fragments, by using the formula appended below:
pmol = (weight in ng) X 1000 / (base pairs X 650 Daltons)
Also, the protocol suggested an optimum range of vector concentration of 50 ng-100 ng. Let's say if I were to use a vector of size 2000 bp, the pmol range obtained for 50 ng - 100 ng is 0.038 - 0.076 pmol, a range far from 0.2 - 1 pmol. Would appreciate if anyone could provide me suggestions and advice on this matter. Thank you.
Hi there,
According to NEB protocol for 4-6 fragments assembly into a vector, the range of 0.2-1pmol is the total amount of DNA present in the assembly mix (ie vector and fragments together). The range of 50ng-100ng is about the mass of vector alone present in the mix.
This doesn't need to be precise. The technique is very tolerant to deviations. I've done scores of different Gibson reactions and had all kinds of problems. Not precisely calculating the masses was never one of them :) It will generally either work really well, with most colonies being correct over a broad range of conditions; Or it won't work at all no matter what you do.
If insert and vector are approximately the same size, use 50 ng vector and 150 ng insert.
If they are different sizes, correct by the ratio of their length, so that you add less mass of the smaller molecule (i.e. keeping the amount constant at 3x insert excess).
If you don't get any correct colonies, it's not the fault of your precise DNA ratios. It's the fault of picking GC rich overlaps that won't work, or some other problem.
Dear Dominique,
Thank you for your explanation. I had misunderstood on the concentration part from NEB's protocol. Thank you again for the clarification.
Dear John,
Thank you for your reply. I have not performed any Gibson cloning yet and will be doing it for the first time real soon. From your experience, do I have to set the minimum concentration of my vector (50 ng) as a benchmark regardless of the size of vector every time I perform a Gibson cloning?
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