Eckdahl: Created page with "Golden Gate Assembly Single Molecule Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL 10X Promega Ligase Buffer :7 µL dH₂O :0.5 µL Restrictio..."

2015-06-01T19:32:53Z

Created page with "Golden Gate Assembly Single Molecule Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL 10X Promega Ligase Buffer :7 µL dH<sub>2</sub>O :0.5 µL Restrictio..."

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Golden Gate Assembly Single Molecule Protocol
GGA mixture contains:

:1 µL (50 ng) Plasmid

:1 µL 10X Promega Ligase Buffer

:7 µL dH<sub>2</sub>O

:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)

: 0.5 µL T4 DNA Ligase to the mixture

:10 µL 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.

Golden Gate Assembly Single Molecule Protocol - Revision history

Eckdahl: Created page with "Golden Gate Assembly Single Molecule Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL 10X Promega Ligase Buffer :7 µL dH2O :0.5 µL Restrictio..."

Eckdahl: Created page with "Golden Gate Assembly Single Molecule Protocol GGA mixture contains: :1 µL (50 ng) Plasmid :1 µL 10X Promega Ligase Buffer :7 µL dH₂O :0.5 µL Restrictio..."