I am a first time user and I obtained expression both in Induced and uninduced (even more expression) using BL21 (DE3) after culturing in LB medium and inducing with 1 mM IPTG. It looks very strange, however, the proteins seem to appear at the right size.
Any explanation?
One of my labmates has tried before and he expressed it successfully. You said something looks strange, are you mentioning the protein? Can you explain more on the "strange" term?
Hi Yen, What I find strange is the fact that there is overexpression in the uninduced as well, I am pretty sure no IPTG was added.
Thank you to all for your contributions, just found out it is normal to see overexpression in IPTG uninduced. The inducer in pCold is the lower temperature. IPTG is normally used to increase the amount of T7 RNA polymerase in the cell, the T7 RNA polymerase will then bind to the T7 promoter to transcribe the gene of interest. pCold-I does not have a T7 promoter , however the manufacturer of pCold-I still recommend to use IPTG.
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