Hello!
When trying to transform E. coli DH10B or TOP10 with a large plasmid, like more than 65k bp, what do you think is the best time for the cells to be let recovering during the recovery time (following electroporation and before spreading the cells onto the agar plate)?
Most protocols says to let the cells recovering for 1 hour at 37ºC, but I am thinking that maybe with a large plasmid, this time should be extended? Have you experienced something like extending the recovery time for 2 or 3 hours, and then plating the cells, increases the transformation efficiency?
Thank you for your help and insights!
Best.
when I tested it there was no difference. the large size should not affect the marker expression and the recovery phase is for marker expression hence it should not make a difference if you are using a standard marker. if you are working with a new marker or you adjusted the promoter of your marker it might be beneficial to extend the recovery time. The limiting step for large plasmid is uptake, however, in your case a 65kb is compatible with electroporation.
Size should be irrelevant as Hanna Alalam mentions, since plasmid size won't affect gene expression of the resistance element.
The main reason NOT to extend recovery time is that you start getting cell division cycles so that not every resistant clone is unique but rather they are siblings. So if there was only 1 actual transformant but you gave enough recovery time for 3 generations then you might have 8 colonies but all would be identical clones. Two hours recovery would be ample time for a number of generations.
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