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Team:Paris Saclay/gibson
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= '''Gibson Assembly''' = | = '''Gibson Assembly''' = | ||
| - | + | Isothermal Assembly | |
| - | + | Isothermal Assembly works by combining a cocktail of exouclease, polymerase, an ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT over lap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50°C. | |
| - | + | To perform isothermal assembly: | |
| - | 3. Add | + | 1. PCR up your fragments of choice, and gel purify. Also gel purify the cut vector |
| + | |||
| + | 2. Not exceeding a total volume of 5 µL, in a PCR tube, combine fragments at equal molecular ration [e.g. amount fragment 1 = 100 ng * (fragment size 1 / fragment size 2); amount fragment 2 = 100 ng* (fragment size 2 / fragment size 1)... etc.]. If required, bring to 5 µL with ddH2O. We recommend using approximately 100 ng of plasmid backbone. | ||
| + | |||
| + | |||
| + | 3. Add the combined fragments (5 µL) to one Isothermal Assembly reaction aliquot (15 µL) and mix by pipetting (20 µL total). | ||
| + | |||
| + | 4. Place mix at 50 °C for 60 min. | ||
| + | |||
| + | 5. (optional for chem. transformation)Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 µL of ddH2O. [or simply dialyse] | ||
| + | |||
| + | 6. Transform with 10 µL of assembly mix | ||
| - | |||
Revision as of 17:37, 3 October 2013
Gibson Assembly
Isothermal Assembly
Isothermal Assembly works by combining a cocktail of exouclease, polymerase, an ligase to fuse dsDNA fragments with sufficiently (20-120 bp) homologous ends. It leaves no "scar" behind, i.e. you can expect your product to contain the EXACT over lap sequence. The reaction may work with shorter ends (e.g. 15 bp), so long as the annealing temperature is higher than 50°C.
To perform isothermal assembly:
1. PCR up your fragments of choice, and gel purify. Also gel purify the cut vector
2. Not exceeding a total volume of 5 µL, in a PCR tube, combine fragments at equal molecular ration [e.g. amount fragment 1 = 100 ng * (fragment size 1 / fragment size 2); amount fragment 2 = 100 ng* (fragment size 2 / fragment size 1)... etc.]. If required, bring to 5 µL with ddH2O. We recommend using approximately 100 ng of plasmid backbone.
3. Add the combined fragments (5 µL) to one Isothermal Assembly reaction aliquot (15 µL) and mix by pipetting (20 µL total).
4. Place mix at 50 °C for 60 min.
5. (optional for chem. transformation)Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 µL of ddH2O. [or simply dialyse]
6. Transform with 10 µL of assembly mix
