Electroporation

Kyle Legate via methods%40net.bio.net (by legatekBLAH from hotmail.com)
Sun Feb 8 05:16:13 EST 2009


Christian Praetorius wrote:

>> for a little longer at 37C seems to work just as well as 42C. Saves 
>>from paranoia in the lab about someone "absolutely needing" 42C
>> bath NOW :-)
> 
> :-)
> The heat shocking is something religious anyway. Ask two and they will
> tell you at least three different times for the heat shock. I found
> anything between 30sec and 2min working.
> 
Heat shock is not even necessary in most cases. It depends on the 
strain, mostly. In my hands, SURE cells require a heat shock but 
DH5alpha do not. Since I use DH5alpha these days for cloning I only used 
heat shock for a particularly troublesome blunt ligation that was rather 
inefficient (heat shock does raise the transformation efficiency by 
about an order of magnitude, but since my competent cells are often 
~10^9 I don't need it). My protocol:

1. Incubate DNA mix (ligation, supercoil, etc) with E. coli 5-10 minutes 
on ice.
2. Add 10X vol room temp LB and directly plate ~30 uL (for supercoil) or 
100 uL (for ligation).
3. ???
4. Profit!

Kyle


More information about the Methods mailing list