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
