Gibson Assembly, developed by Dr. Daniel Gibson and his colleagues at the J. Craig Vendor Institute is an effective method for the assembly of multiple DNA fragments. This is accomplished in a single tube isothermal reaction with Gibson Assembly Master Mix. The method utilizes adjacent DNA fragments with complementary ends which can be added for example by PCR. Then, the overlapping fragments are added the Gibson Assembly Master Mix and incubated for 15 minutes to one hour at 50 degrees Celsius. During the incubation, the Master Mix's three enzymes activities set to work on the fragments.
First, a five prime to three prime exonuclease activity creates single stranded three prime overhangs. These complementary sequences then anneal, creating the double stranded DNA of interest. DNA polymerase then extends the three prime ends, filling in the gaps and DNA ligase seals the remaining nicks. The end result is a fully sealed double stranded DNA molecule that can serve as template for PCR, RCA or a variety of other molecular biology applications including direct transformation.
Gibson Assembly has been successfully used to reliably join up to six DNA fragments into a single molecule. This remarkably straightforward and powerful techniques makes quick work of large multi-fragment assemblies but it is also useful for more routine applications such as cloning. The Gibson Assembly cloning kit which includes both Gibson Assembly Master Mix and NEB® 5-alpha competent cells, has been optimized for efficient assembly and cloning of multiple fragments into any vector in just over an hour.
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