Difference between revisions of "Golden Gate Assembly for Bio113"
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'''GGA mixture contains:'''<br> | '''GGA mixture contains:'''<br> | ||
| − | * 1 µL (40 nM) plasmid containing your receiving plasmid (pClone | + | * 1 µL (40 nM) plasmid containing your receiving plasmid (pClone)<br> |
* 6 µL dH<sub>2</sub>O <br> | * 6 µL dH<sub>2</sub>O <br> | ||
* 1 µL 10X Promega Ligase Buffer<br> | * 1 µL 10X Promega Ligase Buffer<br> | ||
| − | * 0.5 µL Bsa I high fidelity ( | + | * 0.5 µL Bsa I high fidelity (HFv2) restriction enzyme<br> |
* <u>0.5 µL T4 DNA ligase from Promega</u><br> | * <u>0.5 µL T4 DNA ligase from Promega</u><br> | ||
* 9 µL final volume <br> <br> | * 9 µL final volume <br> <br> | ||
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Turn on the thermocycler. Put '''both''' of your tubes into the machine. <br> | Turn on the thermocycler. Put '''both''' of your tubes into the machine. <br> | ||
Program it for the following cylces: <br> | Program it for the following cylces: <br> | ||
| − | * 20 cycles of 37C for 1 minute | + | * 20 cycles of 37C for 1.5 minute and 16C for 1.5 minute <br> |
| − | * | + | * 37C for 3 minutes<br> |
* 22C holding temperature <br> | * 22C holding temperature <br> | ||
| + | * heated lid on | ||
| + | |||
This DNA ligation is ready for transformation. <br> <br> | This DNA ligation is ready for transformation. <br> <br> | ||
Latest revision as of 14:02, 5 February 2020
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)
- 6 µL dH2O
- 1 µL 10X Promega Ligase Buffer
- 0.5 µL Bsa I high fidelity (HFv2) 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 1.5 minute and 16C for 1.5 minute
- 37C for 3 minutes
- 22C holding temperature
- heated lid on
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.
