MWSU protocols

Purification of DNA Golden Gate Assembly Protocol for BsmB1

GGA mixture contains:

1 µL (50 ng) Plasmid 1 µL Promoter 1 µL 10X Promega Ligase Buffer 6 µL dH2O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme 9.5 µL volume

Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture.

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 BsmB1

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.

Isolation of Genomic DNA from Bacteria

PCR

Standard PCR

Resuspending Oligos

Colony PCR

Recombinant DNA Production

Pouring an Agarose Gel

BioBrick Digestions for Fragment and Vector Preparation

Fragment Purification

Gibson Assembly

Direct Synthesis with Overlapping Oligos

Annealing Oligos for Cloning