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Team:Groningen/Template/MODULE/Notebook/protocols/gibsonassembly
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| - | Gibson assembly is a nice way to join multiple DNA fragments without creating a scar between the fragments. For the assembly primers are designed in such a way that all the fragments have a 40 bp overlap at their ligation point. Then, a PCR is performed on all the fragments to create the overhang. Finally, the assembly is done | + | Gibson assembly is a nice way to join multiple DNA fragments without creating a scar between the fragments, see figure 1. For the assembly primers are designed in such a way that all the fragments have a 40 bp overlap at their ligation point. Then, a PCR is performed on all the fragments to create the overhang (1). Finally, the assembly is done. The assembly uses a T5 exonuclease to create single-stranded 3' overhangs that cause annealing of complementary fragments. (2) Then a polymerase in the same mixture fills in the gaps between each fragment and a DNA ligase seals the nicks. (3) The ligated fragments can then directly be used to transform the bacterium of your choice. (4) |
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Latest revision as of 01:30, 18 October 2014
Gibson assembly
Gibson assembly is a nice way to join multiple DNA fragments without creating a scar between the fragments, see figure 1. For the assembly primers are designed in such a way that all the fragments have a 40 bp overlap at their ligation point. Then, a PCR is performed on all the fragments to create the overhang (1). Finally, the assembly is done. The assembly uses a T5 exonuclease to create single-stranded 3' overhangs that cause annealing of complementary fragments. (2) Then a polymerase in the same mixture fills in the gaps between each fragment and a DNA ligase seals the nicks. (3) The ligated fragments can then directly be used to transform the bacterium of your choice. (4)
Figure 1:
Gibson assembly
Procedure
A PCR is done on the fragments with the primers that were designed for the assembly. Then the assembly is performed in the 2x Gibson Assembly Master Mix from NEB. The fragments are added to 10 μl of this mix, 0.25 pmol each, giving a total volume of 20 μl. The mix is then incubated at 50 °C for 1 hour.
