Difference between revisions of "Golden Gate Assembly Protocol for BsmB1"
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GGA mixture contains: | GGA mixture contains: | ||
| − | :1 µL (50 ng) | + | :1 µL (50 ng) receiving plasmid |
| − | :1 µL promoter or other insert | + | :1 µL promoter or other insert at X ng/µL (to determine X ng of insert = (bp insert) (50 ng linearized plasmid-) ÷ (size of entire plasmid in bp) |
| − | :1 µL 10X | + | :1 µL 10X NEB Ligase Buffer |
:6 µL dH<sub>2</sub>O | :6 µL dH<sub>2</sub>O | ||
| Line 12: | Line 12: | ||
:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI) | :0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI) | ||
| − | : 0.5 µL T4 DNA Ligase to the mixture | + | : 0.5 µL T4 DNA Ligase from NEB to the mixture |
:10 µL total volume | :10 µL total volume | ||
| − | Turn on the | + | Turn on the thermocycler machine. Put tube into machine. Program it for the following cycles: |
| − | :• 20 cycles | + | :• 20 cycles of |
| − | :• | + | :• 37°C for 1 minute |
| − | :• | + | :• 16°C for 1 minute |
| + | after 20 cycles: | ||
| − | :• | + | :• 37°C for 15 minutes |
:• 22°C holding temperature | :• 22°C holding temperature | ||
Revision as of 16:39, 8 June 2016
Golden Gate Assembly Protocol GGA mixture contains:
- 1 µL (50 ng) receiving plasmid
- 1 µL promoter or other insert at X ng/µL (to determine X ng of insert = (bp insert) (50 ng linearized plasmid-) ÷ (size of entire plasmid in bp)
- 1 µL 10X NEB Ligase Buffer
- 6 µL dH2O
- 0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)
- 0.5 µL T4 DNA Ligase from NEB to the mixture
- 10 µL total volume
Turn on the thermocycler machine. Put tube into machine. Program it for the following cycles:
- • 20 cycles of
- • 37°C for 1 minute
- • 16°C for 1 minute
after 20 cycles:
- • 37°C for 15 minutes
- • 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.
