problem in expressing A DNA binding protein
Posted 16 February 2012 - 05:28 AM
i have exprienced some problem in expressing a DNA binding protein in Bl21(DE3) cells.
i have checked my gene sequence in the plasmid after ligation by sequencing.it is correct n in correct reading frame.
i have tried many things. given IPTG induction at different O.D.s like 0.6, 0.8, 1.0 even. induced the protein for different time periods after induction i.e. for 4 hrs, 6 hrs. tried expressing at 37 degree and 25 degree(overnight).
even after trying all i get only 1mg of my protein from 1litre of the culture.
a wierd thing that i have noticed is that my Bl21 cells transformed with plasmid carrying my gene of interest reach the O.D. of 0.6 only after about 4 hours which i think is about double the time taken by Bl21 cells without the plasmid.
so i have two questions:
1) how can i increase the expression of my protein.
2) why are the bl21 cells growing so slowly after transformation with my plasmid.
Posted 06 April 2012 - 12:03 AM
Posted 28 May 2012 - 10:21 PM
so what else did u try zhengming??
Edited by gurmeet kaur, 28 May 2012 - 10:22 PM.
Posted 30 May 2012 - 02:49 AM
Posted 30 May 2012 - 04:25 AM
Posted 31 May 2012 - 09:29 AM
Posted 12 June 2012 - 11:57 AM
Also, try baffled erlenmeyer flasks- the dents in the flask does a better job aerating cultures and cells grow and express better. I've seen big difference between cultures grown in regular and baffled flasks.
I've heard good things about the Lucigen high comp cells- good for toxic proteins that kill your culture