Transforming chemically competent cells

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*'''[[User:Hugh Kingston|hugh kingston]] 09:46, 4 February 2008 (CST)''':I tried a few variations on this protocol, and found
*'''[[User:Hugh Kingston|hugh kingston]] 09:46, 4 February 2008 (CST)''':I tried a few variations on this protocol, and found
*using SOC instead of LB + 20mM glucose increased efficiency 3 fold
*using SOC instead of LB + 20mM glucose increased efficiency 3 fold
-
*heat shock of either 42°C 45s increased efficiency 15-20 fold
+
*heat shock of 42°C 45s increased efficiency 15-20 fold
[[Category:Protocol]]
[[Category:Protocol]]
[[Category:Escherichia coli]]
[[Category:Escherichia coli]]

Revision as of 10:46, 4 February 2008

Also see Preparing chemically competent cells

Method

  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
    • Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
  3. Let sit for 30 minutes on ice.
    • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 30 seconds at 42oC.
    • Note: According to the original TSS paper and qualitative experience (JM), this step is completely optional and may actually reduce transformation efficiency.
  5. Incubate cells on ice for 2 min.
  6. Add 1 mL SOC (2XYT and LB are also suitable, original paper suggests LB + 20mM glucose) at room temp.
  7. Incubate for 1 hour at 37oC on shaker.
    • Note: Can also save some time here by reducing incubation to ~45 min.
  8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
  9. Grow overnight at 37 °C.
  10. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
  • hugh kingston 09:46, 4 February 2008 (CST):I tried a few variations on this protocol, and found
  • using SOC instead of LB + 20mM glucose increased efficiency 3 fold
  • heat shock of 42°C 45s increased efficiency 15-20 fold
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