During Transformation, before heat shock at 42 degrees for 60-90 sec we keep the plasmid and bacterial cell mixture on ice for 30 min. Will some one help me why we do that? Thanks in advance
Keeping the cell temperature low will keep the temperature margin high, therefore increasing the effectiveness of "heat shock".
That is to increase the 'competence' of the bacterial cells, so that the plasmid can enter the cells more readily because of increased pore size resulted from the greater heat shock.
from what I know is that incubating on ice for 30 mins is to stabilise the bacteria membrane as it increases the interaction between Ca ions and negatively charged components such as DNA and bacteria surface which is important in membrane permeability.
then the heat shock is to improve the uptake of the DNA into the cells as it changes the fluidity of the membrane and increase pore sizes.
last (but I'm not sure why) is the 2-3 mins incubation on ice is to increase the chance as well of the plasmid DNA to enter the cell!!
Since it stabilizes the plasmid with the competent cell. 30 min is enough for this purpose. At low temperature the molecular vibration must be very low and it enhances retention of bound plasmid. It is okay if you extend this time to 1 hour, transformation will happen.
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