"
Team:Cambridge/Protocols/Gibson Assembly
From 2011.igem.org
(Difference between revisions)
(→Practice) |
(→Practice) |
||
| Line 10: | Line 10: | ||
Master Mix for Gibson Assembly | Master Mix for Gibson Assembly | ||
{| border="1px" | {| border="1px" | ||
| - | ! scope="col" width=" | + | ! scope="col" width="220" |Reagent |
| - | ! scope="col" width="150" |Volume (µl) | + | ! scope="col" width="150" style="text-align:center;" |Volume (µl) |
|- | |- | ||
|Taq ligase (40u/µl) | |Taq ligase (40u/µl) | ||
Revision as of 20:38, 20 September 2011
Loading...
Gibson Assembly
Theory
Gibson Assembly as a scar free method of DNA recombination that is highly efficient and readily copes with combination of multiple DNA fragments at once.
Practice
Master Mix for Gibson Assembly
| Reagent | Volume (µl) |
|---|---|
| Taq ligase (40u/µl) | 50 |
| 5x isothermal buffer | 100 |
| T5 exonuclease (1u/µl) | 2 |
| Phusion polymerase (2u/µl) | 6.25 |
| Nuclease-free water | 216.75 |
| Total | 375 |
Master Mix is 1.33x concentrated
DNA and Gibson Master Mix should be combined with a volumetric ration of 1:3 in a PCR tube. The total volume can be 20-50µl.
Set thermocycler containing the PCR tubes to 50 degrees C for 1 hour.
Safety
