The protocol calls for 42 degree celcius. Would incubation at 45 degree celcius affect the transformation efficiency?
Higher temperatures have a negative impact on transformation efficiency (probably because fewer cells survive the increased heat shock) in my experience.
Regards,
Christian
You are talking about incubation and that is function of time if you incubate your cells for 30 second it not the same as 1 minute. For 30 seconds at 45 C° the majority of your cells are a live and the transformation efficiency will decrease a little but not significantly
At longer time the cells are dead and the efficiency is impacted.
Dear Mohd,
As commented before, competent bacteria might not survive the heat shock when temperatures are high, but `i think that 45 C isn't high enough to kill your transformation. I guess you post this because you haven't got any colony. I'm doing my transformations at ranges between 42 to 44 C and they work well. I'm using homemade ultracompetent bacteria (Inue protocol) derived from DH5a. If you are using 45 C just try to reduce the heat shock to no more than 1 min. Have in mind that optimal temperature and duration for the heat shock also depends on the volume solution in which your bacteria and DNA are mixed.
Good luck!
Thanks everyone! I manage to get some colonies and the transformation control did not show any significant changes. I did the transformation for 20 sec only when I realized I dip my cells in 45 C.
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