the vector for stable transfection
Posted 27 February 2004 - 05:39 AM
so i do not know if these clones are true positive clones (since there are no fluorescence)and i don't know how to do now. should i throw away these clones and do another transfection ? Maybe i can do some western blots to know if the gene has integrated into the genome and began to express ? can i isolate the genomic DNA and do some PCR to confirm this?
some people told me that pEGFPC3 is not very suitable for stable transfection and they said that pcDNA3.1 is better. Is it ture?
Posted 08 March 2004 - 09:26 PM
you can definitely do a pcr to confirm the presence of your DNA in your transfectants, you'd have to use plasmid specific primers, so that you don't get a signal from anything cross reacting in the genome. Or try one plasmid specific and one gene specific primer. You can use sequencing primers most of the time (I'm not sure if your vector is routinely used for cloning?), just check that they are compatible with each other (ie have similar Tms).
As far as I know it doesn't matter what vector you use for transfection, but one thing you can if your transfectants turn out to be false positives, is to increase you concentration of G418 when first isolating resistants, a response curve might help, concentrations required to kill different cell lines is different.
Posted 01 April 2004 - 02:05 AM
I am also venturing into this area and would be happy to help and exchange pointers.
Posted 25 October 2004 - 02:46 AM
GFP protein doesn't last long, some people say it lasts 10 days, others say one month. Please check your gene expression first before you throw away these colonies.