What happens if you add IPTG directly when you expand your training

It can also induce proteins, and I have encountered a similar situation in experiments before.

The reason why IPTG is added in the induction stage is to minimize the impact of a large number of expressed proteins (usually the proteins that need to be expressed are also not needed by the engineered bacteria) on the growth of the cell, so as to facilitate the early arrival of the required cell concentration, which is conducive to the expression of a large number of proteins in the induction stage. Generally speaking, the problem mentioned by the subject may have an impact on the growth rate of the cell, and some of the more sensitive proteins may form inclusion bodies.

Or the concentration changes, but the protein expression should be fine.

However, it is recommended that it is best to test when extracting protein, and if the concentration or solubility does not meet the requirements, discard it as soon as possible, and re-induce it so as not to affect the experiment.

Adding IPTG directly at the time of expansion can cause some problems. IPTG is an inducer that can induce bacteria to express exogenous proteins, but its addition needs to be carried out during the induction phase, not during the propagation phase.

The addition of IPTG at the time of propagation may affect the growth rate of the cell, and some of the more sensitive proteins may form inclusion bodies or change concentrations. If the concentration or solubility is not as desired, it may affect the experimental results. Therefore, it is recommended to add IPTG during the induction phase to ensure the efficiency and stability of protein expression.

Generally, it is a prepared Amp+LB plate, and when coating the plate, add X-GAL 40UL (20mg ml) and IPTG 4UL (200mg ml) to the bacterial solution, and then mix the plate. It can also be applied to the plate first before applying the bacteria.

When preparing the medium, X-gal and IPTG are generally not added directly, which does have the problem of waste, because the bacteria only grow on the surface of the plate.

Escherichia coli culture expression is generally done after the previous molecular cloning work has been completed, and at this time, the monoclonal bacteria picked out from the plate will be first transferred to a relatively small volume of medium, which is generally about 100ml. Because the LB medium used is less nutritious, it is necessary to shake the flask overnight to allow the bacteria to grow to a sufficient amount. It is then transferred to a larger volume of medium and re-incubated.

When the volume of bacteria grows rapidly in proportion to the logarithmic phase, it can be induced with IPTG.

Therefore, it is said that the purpose of shaking the bacteria overnight is to allow the bacteria to grow in large quantities, and the transfer is to expand the culture volume in order to obtain more metabolites.

I have not done capsular erythrocytes, but I know that IPTG has a certain toxicity to cells, so that the cells almost do not replicate, but express and produce a large number of proteins, so the number of bacteria in the medium will not increase much, but it is not clear whether it will become clear, and it may be an error in judgment.

IPTG is called isopropyl--D-thiogalactoside in Chinese, and is usually prepared with water or 20% stock solution.

It can be stored at -20 degrees Celsius for a long time. But we can also use it at 4 degrees for a few weeks, which is relatively stable.

Hope it helps!

Personally, I feel that it is contaminated by bacteriophages. When I was doing plasmid amplification, I also encountered a decrease in OD, but the OD rose very slowly and the highest was reached. What protein do you express?

What would happen if the people of our planet grew unlimitedly; Inhibitions are released between the strains, and after 10 hours, some of the strains are eliminated, and the remaining ones are fully absorbed by the nutrients.

Darwin's Theory of Evolution. Half-life was restored again.

Add some water, or liquid to try, I can't guarantee it. Give a point!!

It is.. If your eluate is 1L, then add sodium chloride.

The density of the aqueous solution is 1. So... Got it.