HELP! no expression in BL21 - (Nov/15/2006 )
Dear all,
Pls give me some advices!
I can't get the expression from pET30a vector in BL21(DE3) lysS cell. I tried different temps (30 degree and 37 degree), different concentration of IPTG (0.5mM, 1mM and 2mM) and different induction times (1hr, 2hr, 4hr and o/n).
I checked the sequence is in frame and the protein should not be toxic (since cell no was increased after induction).
What's going on? What can I do now??? Anyone can help me? 
THx so much.
Percy
hi
i think u need to do more variations in conditions
try more different temp like 20,25, and sometimes even 40 ( though very little chance,at 40 keep for lesser hours like one hour or less than five )
also try more variation in time from 2 hr, 3 hr, 5hr , 7 hr and 9 hr
if the culture is not growing at 20 even try 18 overnight
also IPTG too variation -- 0.01,0.03.....etc at different temp
also 0.1 0.3 ...upto 0.8 at different temp
if not lesser concentration try larger concnetration s
all the best
hope this helps
try reading the manuals trouble shooting part it might give u more idea
laxmi
PLEASE DOWNLOAD THIS PDF AND READ IT , IT MAY HELP.
EXPRESSION IN BL21
http://www.biomol.de/dateien/infos_nr353.pdf
i think u need to do more variations in conditions
try more different temp like 20,25, and sometimes even 40 ( though very little chance,at 40 keep for lesser hours like one hour or less than five )
also try more variation in time from 2 hr, 3 hr, 5hr , 7 hr and 9 hr
if the culture is not growing at 20 even try 18 overnight
also IPTG too variation -- 0.01,0.03.....etc at different temp
also 0.1 0.3 ...upto 0.8 at different temp
if not lesser concentration try larger concnetration s
all the best
hope this helps
try reading the manuals trouble shooting part it might give u more idea
laxmi
Thx so much, i must try them.
EXPRESSION IN BL21
http://www.biomol.de/dateien/infos_nr353.pdf
Thank you. I hope I can get some info to solve my problem.
PLEASE DOWNLOAD THIS PDF AND READ IT , IT MAY HELP.
EXPRESSION IN BL21
http://www.biomol.de/dateien/infos_nr353.pdf
Thank you. I hope I can get some info to solve my problem.
i think the exogenous protein will be harm to host(BL21 component cells), u can try add Glc to medium or induce at 16 temp overnight.
Pls give me some advices!
I can't get the expression from pET30a vector in BL21(DE3) lysS cell. I tried different temps (30 degree and 37 degree), different concentration of IPTG (0.5mM, 1mM and 2mM) and different induction times (1hr, 2hr, 4hr and o/n).
I checked the sequence is in frame and the protein should not be toxic (since cell no was increased after induction).
What's going on? What can I do now??? Anyone can help me?
THx so much.
Percy
Did u check for the codons in your sequence. Try BL21 (RIL) codon plus cells or some other cells.
deeps
did you try western? sometimes it is hard to detect proteins on PAGE gel. transfer and check by western. good luck!