Difference between revisions of "Golden Gate Assembly Protocol"

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Line 8: Line 8:
 
:1 µL 10X Promega Ligase Buffer  
 
:1 µL 10X Promega Ligase Buffer  
  
:6 µL dH<sub>2</sub>O  
+
:6.9 µL dH<sub>2</sub>O  
  
 
:0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)
 
: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
  
:10 µL total volume  
+
:10.9 µL total volume  
  
  

Latest revision as of 14:15, 9 February 2017

Golden Gate Assembly Protocol GGA mixture contains:

1 µL (20 ng) Plasmid
1 µL promoter or other insert (1:1 molar ratio to Plasmid DNA)
1 µL 10X Promega Ligase Buffer
6.9 µL dH2O
0.5 µL Restriction enzyme (BsmBI-HF, BsaI-HF, or BbsI)
0.5 µL T4 DNA Ligase to the mixture
10.9 µL total volume


Turn on the thermal cycler machine. Put tube into machine. For BsaI or BbsI GGA, program it for the following cycles:

• 30 cycles
• 37°C for 1 minute
• 16°C for 1 minute
• 37°C for 15 minutes (after 30 cycles)
• 10°C holding temperature


Turn on the thermal cycler machine. Put tube into machine. For BsmBI GGA, program it for the following cycles:

• 20 cycles
• 55°C for 10 minutes
• 37°C for 1 minute
• 16°C for 1 minute
• 10°C holding temperature

After GGA, the DNA 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.