Gibson Assembly® Master Mix – Assembly (E2611)
New England Biolabs
Abstract
This protocol explains methods for the Gibson Assembly using the Gibson Assembly® Master Mix (E2611).
Before start
Steps
Set up the following reaction On ice:
| A | B | C | D |
|---|---|---|---|
| Recommended Amount of Fragments Used for Assembly | |||
| 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control** | |
| Total Amount of Fragments | 0.02-0.5 pmols*X ul | 0.2-1 pmols*X ul | 10 ul |
| Gibson Assembly Master Mix (2X) | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μl*** | 20 μl*** | 20 μl |
*Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.**Control reagents are provided for 5 experiments.***If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
Incubate samples in a thermocycler at 50°C for 0h 15m 0s when 2 or 3 fragments are being assembled or 1h 0m 0s when 4-6 fragments are being assembled.
Store samples on ice or at -20°C for subsequent transformation.
