IPTG induction time - (Jul/10/2006 )
goodmorning all
i want anyone to check my protocol :
i do an ON culture in 10 ml LB media then in the morning the starting OD is 2 mainly then i do 1:20 diltuion in 100 ml new media with starting OD 0.1
after an hour in 37 shaking my OD is usully 0.4 or 0.5 then i induce with IPTG for another one hour usully the next OD is 1.6 but today i found it 1.02 and another sample was 0.98 ????
i put it again for another one hour to be checked later...
do u have any explanation what happened this time?
or is it normal to get this OD? is this realted somehow to the IPTG induction?
thankx in advance
its me again after one hour
OD is 1.03?????
stupied question to ask if i am using an 500 ml flask to grow 100 ml culture should i leave it without a cover, i used alominum foil cover , the one used in autoclaving and make it loose?
may be this is the reseon?
OD is 1.03?????
stupied question to ask if i am using an 500 ml flask to grow 100 ml culture should i leave it without a cover, i used alominum foil cover , the one used in autoclaving and make it loose?
may be this is the reseon?
First of all, inducing with a loose foil cover is fine.
Next, the difference in OD after one hr could simply be due to a slight difference in the OD at the start of the day. Alternately, sometimes the construct is toxic to cells. Is this the first time you'er expressed this particular protein? However, I doubt that a difference in IPTG concentration would cause such a difference, unless you really dropped it low, or had it really high.
It's just one of those wonderfully random things about bacteria!
thankx swanny
itsnot the first time to express my protein in E coli thats why i was surprised to get this results and the IPTG concentration is the same 1mM
its the first time to do culture on this scale i mean before i used to make 10 ml ON culture then dilute in 10 ml also .
after the 2 hours i found the OD 1.5 i took samples from one hour and 2 hour after induction culture and i am running SDSPAGE now
i will let u know the results.
thankx
If I understand well, the only things that change are the final volume of culture, and the final OD.
Be careful not to overfill the flask. you must be able to shake enough, and the surface of the culture should be as large as possible. It means use a bis flask. I don't have a flask in front of my eyes right now, but a 500 mL flask is better than a 100 mL flask for a 100 mL culture.
But I guess you already knew it, then... I don't know what advice to give you.
thankx missele for ur reply.
after SDSPAGE gel staining i found my protein was expressed well but i dont know yet the cause of the above sitiuation.
anyway i downloaded some lectures from the net about the growth of bacteria to refresh my mind and i am going to read them tonight.
thankx all.
I suppose in the final analysis, if your protein expresses well, that's the main thing, isn't it? Plenty of people here would love to get a well-expressed protein; if the messages are anything to go on, it's a bit of a rarity...
Like I said, it's just one of those random things about bugs!