Why is BL21 used for protein expression?

BL21(DE3)pLysS Competent Cells and Single-Use BL21(DE3)pLysS Competent Cells allow high-efficiency protein expression of any gene that is under the control of a T7 promoter. The strain carries both the DE3 lysogen and the plasmid pLysS.

How long should you induce with IPTG?

The optimal incubation temperature and time for induction will vary depending on the target protein. We recommend varying induction temperature and time to optimize expression (37°C for 2-4 hours, 30°C for 4-6 hours, 22-25°C for 6-16 hours and 12-15°C overnight using 0.4 mM IPTG).

Can you add too much IPTG?

yes IPTG halts the divison process but enhances protein production. however, if we increase IPTG beyond a limit the divison of bacteria is compromised and which in turn effect the protein machinery of the cells. Yes, a high concentration of IPTG is toxic to the cell.

Why do we need to use BL21 DE3 and not BL21 cells for expression with IPTG?

IPTG is required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7 promoter. BL21(DE3) is suitable for expression from a T7 or T7-lac promoter or promoters recognized by the E.

How long does IPTG last?

IPTG is stable for at least 9 months when stored unopened at –20°C.

Does glucose inhibit IPTG?

with glucose). Figure 2, panel B demonstrates that glu- cose addition did not interfere with IPTG induction of the target protein. In fact, IPTG induction from the pLysS host ap- peared to be enhanced in the presence of glucose.

How do you transform in BL21?

Transformation Protocol for BL21(DE3) Competent Cells (C2527)

(For C2527H) Thaw a tube of BL21(DE3) Competent E.
Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture.
Place the mixture on ice for 30 minutes.
Heat shock at exactly 42°C for exactly 10 seconds.
Place on ice for 5 minutes.