Hej,

I can' find puraxicin resistance in the web  - do you mean puromycin resistance? And if GCV resistance means gancyclovir resistance please explain the rational of your experimentation.

Best,

Guenther

Sorry typos :((

yes puromycin and Ganciclovir.

and am planning to do a co-transfection ? so what controls 

thanks

IA

...I still do not understand the aim of your cotransfection and what you wish to control. What you will get is a mixed population of cells. Some containing only one of each plasmid and some will contain both. If you continue with selection for transgenic cells you will then have to screen for clones that contain our target.

I have done a lot of co-transfection by CaPhos, including transfection of two plasmids that need to get into the same cell in order to recombine and produce virus.  I think the genreal idea is that when the HBSS/DNA is mixed with the CaCl2 solution to form insoluble crystals, both plasmids are part of each precipitated mass and that consequently there is a good rate of each cell getting both plasmids.  I don't think this has been formally demonstrated, but the efficiency of co-transfection seems pretty high.

I agree with Guenther that the need for a control is not obvious.  After you transfect and put the cells under selection by both compounds, only cells receiving both plasmids will grow out.  If you're concerned about transfection efficiency, you might transfect a set of cells in parallel with something easy to measure (GFP, beta-gal, etc).  I'm not sure that a single-plasmid control is very informative.  It could be informative to do the transfection in triplicate and perform Puro and GCV mono-selection on each.  Additionally, I would suggest a mock-transfected set be put under selection so that you know that the Puro/GCV reagents you are using indeed kills the cells.

-DW

Thanks Guenther and David.

I did add GFP control and MOC control though.

Let us see how things make up.

And another question just on a side that if the plasmid you are going to transfect does not contain a selectable marker like antibiotic resistance gene then how could we do section and expansion in that transfection ??

Without a selectable marker, you can't really make a stable cell line.  The plasmids without selectable markers are generally for performing transient transfection.

yes i totally agree but was giving it a try that once transfected then i pick up colonies with GFP reporter gene and then run the assay via western blot to detect the gene of interest in the single cell clone ( 96 well method ).

Ah, I see now.  You are not trying to make a long-term stable, rather the GFP is meant as an indicator that the cell was transfected.  The problem you may run into is that the segregation of the plasmid between the two daughter cells is not controlled, so with every cell division, there is a probability that one of the two cells loses the plasmid.

Yeah i think so.But will see how it goes.