Team:UCLA/Notebook/Interlab Study/18 August 2015
Transformation of Device Ligations using chemically competent BL21(DE3) Mix n' Go E. coli cells
Each ligation was was transformed into chemically competent BL21(DE3) Mix n' Go E. coli cells using the Mix n' Go protocol. Megan Satyadi prepared the cell lines using the Mix n. Go Buffer and Transformation Kit.
Transformation followed this protocol:
| Step | Temperature (degrees Celsius) | Time |
|---|---|---|
| Pre-warm 3 LB Agar + Chloramphenicol (34 ug/uL) plates per ligation. Prewarm 200 uL of SOC media per ligation product. | 37 | 1 hour |
| Thaw 1 50uL of cells per ligation from -80 degree freezer and the ligated products from -20 degree freezer. | On ice (0) | 10 minutes |
| Add 2uL of ligation product per aliquot of cells. Briefly flick to mix DNA into cells. | On ice | |
| Let mixture incubate. | On ice | 5 minutes |
| Transfer cell-DNA mixture to pre-warmed SOC media. Invert rescued media 3-4 times to mix. | RT | |
| (For chloramphenicol) Outgrow the cell-DNA mixture in SOC shaking at 650 RPM. | 37 degrees | 1 hour |
| Spin the cell pellets down at 6000 RPM. Discard the old media. | 4 | 5 minutes |
| Resuspend the cell pellets in 100 uL of fresh SOC media. Gently pipet to mix. | RT | |
| Plate 1:1, 1:10, and 1:100 dilutions of each cell mixture on each of the three prewired plates per transformation. | RT | |
| Use sterile glass beads to spread cells in a figure 8 motion. Discard beads carefully after spread. | RT | |
| Let mixture dry on agar, then invert and incubate overnight. | 37 | 16-22 hours. |
| Observe plates for colony growth. Image and place in the freezer for storage with parafilm. | 4 |