problem in transfection
Posted 24 April 2010 - 09:24 PM
m new in transfection field. i try to transfect my vector plasmid 5+ 3 kb insert total 8kb plasmid into cho cells.i m unable to check wether my protein is transfected or not i also transfect egfp plasmid as contol and m able to see florousrence in control.
i tried to check with flow and blot but not able to see any positive result.
sequence of insert was confirmed by sequencing. i also check expression in dh5 strain , i used cell pellet and checked protein through blot but i m unable to see any protein band in blot ,antibodies were working because i used his taged positive control and use his tagg primary antibody as my protein is also his tag at c turminus.
can i check protein expression in dh5 strain because it is mammalian vector and t7 promoter?
is transfection efficiency matter with the size of vector test vector is 8 kb and egfp vector is 4 kb?
is lipofectamine+ plus reagent is good for large vector?
can i linerize my vector so that ther should not be any frame shift?
how the plasmid integrate in cell genome may be through homologous recombination?
Posted 24 April 2010 - 11:57 PM
Transformation efficiency is indeed influenced by vector size ...the smaller the better the transformation efficiencies. But this means not that your transfection efficiencies drop to zero compared with EGFP-vector that is half the size.
I would consider to check your vector once again for frame-shifts, mutations or things like that.
You are going for stable expression or transient?
Posted 25 April 2010 - 12:52 AM
but even after selection my protein was not expressed on cell surface or in supernatant .
Edited by preet, 25 April 2010 - 12:55 AM.
Posted 25 April 2010 - 01:49 AM
This means that transfection was succesful ...the plasmid has integrated.
After what timespan do you tried to detect your protein? Maybe its already silenced?
Posted 25 April 2010 - 06:24 AM
i checked protein after 72 hr of transient and 72 hrs of stable transfected clones.
Edited by preet, 25 April 2010 - 06:27 AM.