Difference between revisions of "Golden Gate Assembly Protocol"
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(Created page with "Golden Gate Assembly Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL promoter or other insert :1 µL 10X Promega Ligase Buffer :6 µL dH<sub>2</sub>O :0...") |
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:1 µL (50 ng) Plasmid | :1 µL (50 ng) Plasmid | ||
| − | :1 µL promoter or other insert | + | :1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA) |
:1 µL 10X Promega Ligase Buffer | :1 µL 10X Promega Ligase Buffer | ||
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:6 µL dH<sub>2</sub>O | :6 µL dH<sub>2</sub>O | ||
| − | :0.5 µL Restriction enzyme (BsmBI | + | :0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI) |
: 0.5 µL T4 DNA Ligase to the mixture | : 0.5 µL T4 DNA Ligase to the mixture | ||
Revision as of 19:06, 1 March 2016
Golden Gate Assembly Protocol GGA mixture contains:
- 1 µL (50 ng) Plasmid
- 1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)
- 1 µL 10X Promega Ligase Buffer
- 6 µL dH2O
- 0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)
- 0.5 µL T4 DNA Ligase to the mixture
- 10 µL total volume
Turn on the PCR machine. Put tube into machine. Program it for the following cycles:
- • 20 cycles
- • 55°C for 10 minutes
- • 37°C for 1 minute
- • 16°C for 1 minute
- • 22°C holding temperature
This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
Transformation of GGA For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.
