Transforming chemically competent cells (Inoue)

Also see Preparing chemically competent cells (Inoue)

Method

1. Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
2. Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
3. Incubate on ice for 30 minutes
   • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
4. Incubate cells for 30 seconds at 42°C.
5. Incubate cells on ice for 2 min.
6. Add 4 volumes of room temperature SOC (not critical)
7. Incubate for 1 hour at 37°C on shaker.
   • Note: Can also save some time here by reducing incubation to ~45 min.
   • Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
9. Grow overnight at 37°C.

Experimental results

First attempt varied several parameters: incubation time on ice prior to heat shock, heat shock length, addition of DTT at 20mM.

• DTT appeared to have little effect when added during transformation.
• Incubating for 1/2 hour on ice had a positive effect, perhaps 1.5 to 2x efficiency gain.
• Heat shock of 0 or 15 s rather than 30 s reduced efficiency about 8x
• Heat shock at 30 s or 60 s gave approximately similar results. (*Edit: 50s is preferable)

Achieved efficiency was 3 x 10⁷ per microgram. A control transformation with Invitrogen cells was at 1.2 x 10⁸ per microgram.

BioStream version

Following is the Transforming chemically competent cells (Inoue) protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

Transforming chemically competent cells (Inoue) protocol

Source Code

Transforming chemically competent cells (Inoue) protocol - source code