Week 12 Lab: Golden Gate Assembly and CRISPR
In this week’s lab, we were introduced to Golden Gate Assembly and CRISPR. We genetically engineered E. coli to express different combinations of fluorescent proteins!
We started by performing the plasmid purification protocol using the new New England Biolabs kit:
Then, each of us chose a “mix” of fluorescent protein colors and intensities to assemble into our plasmids:
I chose Red, Green, and Blue, all at intensity B (the middle option).
We then prepared the reaction in a PCR tube according to the following table:
| Component | Volume |
|---|---|
| bb | 1 µL |
| f_insert | 1 × N µL (my N = 3) |
| Buffer | 2 µL |
| Digestion Enzyme | 1 µL |
| Ligation Enzyme | 1 µL |
| Water | Add to 20 µL |
We then placed the reaction in the thermocycler for approximately 5.5 hours.
Our amazing TAs continued the experiment the next day and transformed our plasmids into E. coli, so unfortunately I do not have pictures of that part!