"
Team:Groningen/Template/MODULE/Notebook/protocols/gibsonassembly
From 2014.igem.org
(Difference between revisions)
(Created page with "<html> <!--Content module--> <div class="module contentmodule gridcell"> <div class="content"> <div class="wrapper"> <!-- TITLE SNIPPET MANDATORY START--> <div class="title">...") |
|||
| (One intermediate revision not shown) | |||
| Line 19: | Line 19: | ||
<!-- PARAGRAPH SNIPPET MANDATORY START--> | <!-- PARAGRAPH SNIPPET MANDATORY START--> | ||
<div class="text"> | <div class="text"> | ||
| - | 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. The assembly uses a T5 exonuclease to create single-stranded 3' overhangs that cause annealing of complementary fragments. Then a polymerase in the same mixture fills in the gaps between each fragment and a DNA ligase seals the nicks. | + | 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) |
</div> | </div> | ||
<div class="hspacer"> </div> | <div class="hspacer"> </div> | ||
<!-- PARAGRAPH SNIPPET MANDATORY END--> | <!-- PARAGRAPH SNIPPET MANDATORY END--> | ||
| + | |||
| + | |||
| + | <!-- FIGURE SNIPPET OPTIONAL START --> | ||
| + | </html>{{:Team:Groningen/Template/MODULE/newfigure|Figure 1|7/79/Sandrasgibsonasseblyofzo.art.png| | ||
| + | Gibson assembly | ||
| + | }}<html> | ||
| + | <!-- FIGURE SNIPPET OPTIONAL END--> | ||
<!-- SUBTITLE SNIPPET OPTIONAL START--> | <!-- SUBTITLE SNIPPET OPTIONAL START--> | ||
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.
