Use the formula below to calculate the recommended amounts of starting
material for combining DNA fragments with any of our Gibson Assembly® kits or
master mixes:
For example:
First, calculate the number of picomoles of your vector
1.55 * [(25 ng/µL)/ 2700 bp] = pmol / µL
1.55 * X ng/µL / 2000 bp= 0.014352
0.000775 X ng/µL = 0.14352 pmol /µL
X = 18.52 ng
For optimal cloning reactions with the HiFi 1-Step and Ultra
kits, the starting DNA fragments will be at a minimum concentration of 40
ng/µL. DNA concentrations of 20–25 ng/µL are acceptable, but efficiency may be
reduced. For more dilute samples (10–20 ng/µL), we offer the same convenient
assembly procedure with consistent and high cloning efficiencies using the Gibson
Assembly® HiFi HC 1-Step kit.For example:
Assume you have a 2.7 kb vector
at a concentration of 25 ng/µL
and three DNA fragments with insert sizes of 750 bp, 1.2 kb, and 2 kb.
In this example you would want to use the recommended vector to insert ratio of 1:1 for the 1.2 kb and 2 kb insert. For the 750 bp DNA
fragment a 1:5 vector to insert ratio should be used as SGI-DNA
scientist recommend a 5-fold molar excess when using fragments < 1 kb. Assume for your Gibson Assembly® reaction that you will use 25 ng of
vector.
To determine how much of each insert are required you can use the
formula above to calculate the pmol of vector and use that result to calculate
how many nanograms of insert are required to have the appropriate molar ratio
of vector to insert. Details on how to perform this calculation are presented
below
First, calculate the number of picomoles of your vector
1.55 * [(25 ng/µL)/ 2700 bp] = pmol / µL
0.014352 = pmol / µL.
Next, calculate for each fragment, solving for X.
For a 2 kb fragment:
1.55 * X ng/µL / 2000 bp= 0.014352
0.000775 X ng/µL = 0.14352 pmol /µL
X = 18.52 ng
Repeat these steps for each DNA fragment. You should get 34.7 ng for a 750 bp fragment (remember, this is a 5-fold increase) and 11.1 ng for a 1.2 kb fragment.
Sum for the vector and DNA fragments, the total amount of starting material should equal 89.3 ng.
Now, combine the inserts and vector DNA and initiate the assembly reaction.
The Gibson Assembly® HiFi HC 1-Step kit (HC kit) contains
the same components as the HiFi 1-Step kit in a formulation that allows for
even more dilute starting material. The introduction of the HC kit to the
SGI-DNA Gibson Assembly® suite of high-efficiency assembly products offers
users greater flexibility for a wider range of starting DNA concentrations.
With all of our Gibson Assembly® kits and Master
Mixes, cloning relies on homologous overlapping ends between adjacent
fragments. Once overlapping fragments are combined in the proper ratios and volumes
as outlined above, they are combined with Gibson Assembly® reagents and
incubated. During incubation, the Gibson Assembly® reagents mediate the
generation of compatible ends, annealing, extension, and ligation to create a
fully assembled seamless DNA construct.
An overview of joining a single insert with a single
vector with the HiFi 1-Step, HiFi HC 1-Step, and Ultra kits is shown below:
![]() |
Seamless DNA assembly of small amounts of starting material DNA with SGI-DNA Gibson Assembly® kits. |
Visit SGI-DNA's web page to learn more about Gibson Assembly® kits and download an electronic version
of SGI-DNA's Gibson Assembly®
Guide to learn about all the ways you can get #DNAMYWAY.
Gibson
Assembly® is a registered trademark of Synthetic Genomics Inc.
Gibson Assembly US
Patent Nos. 7,776,532, 8,435,736, and 8,968,999.
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