Hi..
Recently I do over expression some gene using plasmid target and transfection in hek 293 cells. My plasmids are flag pcDNA3 and pCMV-myc plasmid. When I did that experiment then I analyze western blot using specific antibody of my target, I got the band in right size. Then I generated over expression (transfected in HEK 293 cells) using the other plasmid (pMX-puro vector) with same target and analyzed using western blot, I could not get band in pmx-puro.
I have already tried to add kodak sequence before start codon but failed again.
Can somebody help me to solve my problem?
Thank you
regards,
Dewi
Dear Dewi, are you sure your pMX plasmid works? There could be two problems:
1- the plasmid does not work
2- the cells are not transfected. In my experience the transfection efficiency depends also on the plasmid sequence. Some plasmids gets into the cell and are expressed much easier than others. I suggest to check for transfection efficiency using a visible marker, such as GFP. You may also consider trying different transfection reagents.
Thank Luca Cirillo
1. There is possibility if my plasmid doesn't work but my friend can make its plasmid with his target, we using same empty vector pMX-puro.
2. I've already make my target (around 140bp) using others empty vector (flag and myc)
I make :
- flag-target
- myc-target
- pMX-target
then transfection using lipofectamin and incubate 24h, analyze western blot using specific antibody.
results showed :
- flag-target : i got the band
- myc-target : i got the band
- pMX-target : no band
Because I should use pMX-puro for rescue the mutation so I should solve it and my target size is quite big.
cheers
Dewi
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