E coli culture - (Jul/17/2006 )
Hi, I got JM83 containing my interested protein. This is inoculated in agar. I will amplify culture to get more proteins because I need more proteins for assay. Who can tell me how to do in detail? Thanks a lot.
To be completely alright, you need to know a little more about the plasmid.
is it inducible?
you should ask to whom gave you the bacteria.
You have first to inoculate 1 mL of LB buffer containing the appropriate antibiotics (it depends on the gene coding resistance to antibiotic carried by the vector. you need to get the information). then, you dilute 1000x your bacteria in fresh medium. you let grow until OD600 reaches 0.5. However some proteins can be toxic for the bacteria, and an other OD600 could be prefered. I can not help you for that. When OD600 reaches 0.5, you might need to induce the expression with IPTG? I can't tell you, depends on the plasmid. The amount of IPTG depends also on the protein toxicity, and the duration of induction also.
If I were you, I would ask to the one who gave you the bacteria. There is no stupid question. Don't be shy.
Thanks. one more question, how can I test OD600? what's the normal value of OD600?
Other lab from which I got the plamid use carbenicillin for culture, can I use ampecillin(we don't have carbenicillin in our lab)?
Hi, I got JM83 containing my interested protein. This is inoculated in agar. I will amplify culture to get more proteins because I need more proteins for assay. Who can tell me how to do in detail? Thanks a lot.
To be completely alright, you need to know a little more about the plasmid.
is it inducible?
you should ask to whom gave you the bacteria.
You have first to inoculate 1 mL of LB buffer containing the appropriate antibiotics (it depends on the gene coding resistance to antibiotic carried by the vector. you need to get the information). then, you dilute 1000x your bacteria in fresh medium. you let grow until OD600 reaches 0.5. However some proteins can be toxic for the bacteria, and an other OD600 could be prefered. I can not help you for that. When OD600 reaches 0.5, you might need to induce the expression with IPTG? I can't tell you, depends on the plasmid. The amount of IPTG depends also on the protein toxicity, and the duration of induction also.
If I were you, I would ask to the one who gave you the bacteria. There is no stupid question. Don't be shy.
yes you can use ampicillin, carbenicillin is an analog of ampicillin.
the resistance is carried by the beta-lactamase gene on the plasmid.
To mesure OD600, you need a spectrophotometer. You can use the one you use to quantify DNA. there should be a button with OD600, or you use any spectrophotometer that you can scale to 600 nm (or something close like 595 nm...). You can use plastic single use cuves.
Use the medium as a blank. the value you will read is proportional to the density of your bacteria.
If your bacteria are too dense, they will start to die, and the protein yield will be low due to proteases release. That's why it's better to induce the expression of the protein when the bacteria are in the exponential growth, and to extract the protein out from a culture that didn't reach the plateau of the growing curve. Usually, it means and OD of 0.5 when read at 600 nm.