"
Team:British Columbia/Protocols/GibsonAssembly
From 2012.igem.org
(Difference between revisions)
| Line 28: | Line 28: | ||
This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook. | This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook. | ||
| - | + | #Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X molar concentration if using inserts of 200 bp or less. Add dH2O up to 10 uL. Set up a positive control using 10uL Gibson Assembly Positive Control. | |
| - | + | #Add 10 uL Gibson Assembly Master Mix to each product. | |
| - | + | #Incubate at 50°C for 1 hour. | |
| - | + | #Using 2uL product, transform into competent cells. Since the Gibson Assembly Master Mix contains PEG, avoid using PEG sensitive competent cells. The provided positive control results in an amp-resistant plasmid. | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
Latest revision as of 01:23, 4 October 2012
Gibson Assembly Protocol
This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook.
- Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X molar concentration if using inserts of 200 bp or less. Add dH2O up to 10 uL. Set up a positive control using 10uL Gibson Assembly Positive Control.
- Add 10 uL Gibson Assembly Master Mix to each product.
- Incubate at 50°C for 1 hour.
- Using 2uL product, transform into competent cells. Since the Gibson Assembly Master Mix contains PEG, avoid using PEG sensitive competent cells. The provided positive control results in an amp-resistant plasmid.
