Transformation of competent cells.
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Renumbered during migration. The source protocol skipped from step 4 straight to step 7, with no steps 5 or 6. Worth confirming nothing was lost between the heat shock and the ice step.
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Materials
- Water bath warmed to 42°C
- Agar plates with appropriate antibiotic, at room temperature
- SOC media, at room temperature
Competent cell choice
| Cells |
Use |
| NEB 5-alpha (NEB #C2987I) |
HiFi assemblies |
| JM109 (Promega #L2001) |
Cloning in pGEM-T Easy Vector (Promega #A1360) |
| DH5-alpha (made in-house) |
Other purposes, e.g. C. elegans Fire Lab plasmids |
Follow the kit protocols for commercial cell transformation.
Protocol (JM109 and DH5-alpha)
- Take competent cells out of -80°C and thaw on ice.
- Add 1–5 μL of plasmid (usually 10 pg – 100 ng) or ligation mixture into ~100 μL of competent cells in a cold 1.5 mL tube. Gently mix by flicking.
- Place the competent cell and DNA mixture on ice for 20–30 minutes.
- Heat-shock in a 42°C water bath for 30–60 seconds; 45 seconds is best. Do not shake.
- Place on ice for 2 minutes.
- Add 900 μL of SOC.
- Incubate at 37°C with shaking (225 rpm) for 45 minutes.
- Plate bacteria on agar plates, typically 3 plates with 100 μL, 200 μL, and 300 μL each. Pipette bacteria onto the plate, add ~10 glass beads, put the lid on, and slowly roll the beads through the liquid and around the entire surface of the plate.
- Dump used beads into a glass bottle with 100% ethanol for later reuse.
- Incubate the plates at 37°C overnight.