Team:DTU-Denmark-2/Project/Other assembly systems

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Revision as of 21:23, 5 September 2011

Other assembly systems

Gibson Assembly

Gibson Assembly is an isothermal, single-reaction method for assembling multiple overlapping DNA molecules, which was developed by Daniel G. Gibson at the J. Craig Venter Institute in 2009.
The assembly system employs 5´-T5 exonuclease, Phusion DNA polymerase, and Taq lig and can be used to assemble both ssDNA and dsDNA. This methode makes it possible to join DNA molecules there are as large as 583kb and clone joined products in ''E. coli'' up to 300kb.

The way it's done

The isothermal One-step The 5´-T5 exonuclease removes the bases from the 5'-end of double strained DNA molecule, leaving a recess in the DNA and resulting in useful ssDNA overhangs. The T5 exonuclease are inactivated during the incubation at 50C. Phusion polymerase and Taq ligase fills the gaps of the annealed complementary ssDNA overhangs and seals the nicks in the end.

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 Gibson Assembly is an isothermal, single-reaction method for assembling multiple overlapping DNA molecules, which was developed by Daniel G. Gibson at the J. Craig Venter Institute in 2009.
 <br>
-The assembly system employs 5´-T5 exonuclease, PhusionDNA polymerase, and Taq lig and can be used to assemble both ssDNA and dsDNA. This methode makes it possible to join DNA molecules there are as large as 583kb and clone joined products in ''E. coli'' up to 300kb.<br><br>
+The assembly system employs 5´-T5 exonuclease, Phusion DNA polymerase, and Taq lig and can be used to assemble both ssDNA and dsDNA. This methode makes it possible to join DNA molecules there are as large as 583kb and clone joined products in ''E. coli'' up to 300kb.<br><br>
 <a name="The way it's done"></a><h4><b>The way it's done</b></h3><br>
-The 5´-T5 exonuclease removes the bases from the 5'-end of double strained DNA molecule, leaving a recess in the DNA and resulting in useful ssDNA overhangs. The T5 exonuclease are inactivated during the incubation at 50C, whereas Phusion polymerase and Taq ligase filled the gaps of the annealed sequence and sealed the nicks.
+The isothermal One-step The 5´-T5 exonuclease removes the bases from the 5'-end of double strained DNA molecule, leaving a recess in the DNA and resulting in useful ssDNA overhangs. The T5 exonuclease are inactivated during the incubation at 50C. Phusion polymerase and Taq ligase fills the gaps of the annealed complementary ssDNA overhangs and seals the nicks in the end.