Golden Gate Assembly for Bio113
Starting with Boiled then Cooled Oligos
- You should have already used the Oligator to design your oligos for your dsDNA control element.
- You should have already calculated how to dilute your boiled and cooled oligos from 5 µM to 40 nM.
Protocol Details for GGA
- You will be given two tubes. One tube will be labeled "X" for eXperimental DNA, the other "N" for the negative control that contains plasmid only. Put your initials on the tubes and return the tubes to ice. These tubes will already contain 9 µL of the GGA Mixture.
- Add 1 µL diluted oligos cooled overnight to the tube labeled X for the experimental DNA.
- Add 1 µL water to the tube labeled N for the negative control"
GGA mixture contains:
- 1 µL (40 nM) plasmid containing your receiving plasmid (pClone, or rClone, or actClone, or repClone)
- 6 µL dH2O
- 1 µL 10X Promega Ligase Buffer
- 0.5 µL Bsa I high fidelity (HF) restriction enzyme
- 0.5 µL T4 DNA ligase from Promega
- 9 µL final volume
Turn on the thermocycler. Put both of your tubes into the machine.
Program it for the following cylces:
- 20 cycles of 37C for 1 minute/16C for 1 minute
- 1 cycle of 37C for 15 minutes
- 22C 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.
This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by Annie Wacker and Malcolm Campbell.