Liao, Xin-hua (NIH/NIDDK) [F] wrote:
> Hi, dear all,
> Since I joined Dicty community, I encountered a lot of technique problems or confusions. Some of them may be not a real problem, and the question may be stupid, but they all bother me for a long time. I put them all here, hope you can help me clarify them.
>
> 1.      Blasticidin selection: for knock out, after transformation for around 5 days under blasticidin selection, I transfer single colonies under medium to 96-well plate. For 96 colonies, only a few of them (usually around 10) can really grow up in the wells again. It looks to me a lot of "fake" colonies survive through the drug selection. If the recombination rate happens to be low, this phenomenon will make the screening very difficult to me. 5ug/ml of blasticidin is used. (This concentration is enough to stop WT cells growing and kill them under shaking condition, though).
>   
this is based on my experience some ax2 cells are slightly resistant to 
Bsr even when we used at 5ug/ml Bsr we got some fake colonies (but not 
90% as you see) but to avoid this we do selection at 10ug/ml Bsr. 
Sometimes longer selection in Bsr is also a solution (may be 2 weeks). 
Based on the number of colonies on your plate you can decide whether to 
gradually increase the drug concentration or select for longer or may be 
combination of both may help resolve your problem.

> 2.      G418 selection: for overexpression, after transformation for around 5 days under G418 selection, I spread single cell on KA plate, and then pick single colony to culture in 12-well plate. A lot of them can not expand in the well anymore. Again, I suspect some WT cells survive through the drug selection. 20ug/ml of G418 is used. Even for the real colonies growing up, sometimes only a few of them express target protein, others do not express but are G418 resistant. This will make me picking a stable overexpression cell line is as difficult as screening a knock-out strain!
>   
again try longer selection (2 weeks) but there are other variables too 
-    getting transformants also varies from gene to gene and vector to 
vector, it may take longer too (like once it took almost a month for me 
to get good % of overexpressor mutant). Also from my experience i would 
say try to keep always in drug selection (until you get good % of 
transformants and later you can keep them growing at low concentration 
of drug) because when you take to KA plate (where there is no drug 
selection) the cells tend to get rid of the vector. So best is pick 
colonies directly from selection plate to 96 well plate.

> 3.      Colonies on KA plate: for picking single colony on KA plate, I found if I pick too much KA bacteria with dicty cells to the plate, then the well surface will be occupied by the bacteria (although there are antibiotics in the medium), and the dicty cells would not eat bacteria to proliferate. Instead, they looks "trapped" individually by the surrounding bacteria, and starve to die after a few days!  If I pick much less bacteria with dicty cells, then dicty cells will expand! Is it possible bacteria secreting something to anti dicty?
>   
to avoid this when you pick colony with bacteria resuspend them in the 
medium so that dicty and bacteria are seperate. But I do not think 
bacteria is still alive in the medium (with antibiotics) to secrete 
something that is anti dicty (but that might be interesting)...however 
again there are variables here too...make sure that when you pick you 
have enough dicty cells in an area because even very low density of 
cells in a given area will tend to die depending on the mutant you are 
working with.

> 4.      Colonies size on KA plate: the colonies sizes are not uniform at all even for cells from the same strain. And the size is also dependent on the physiological stage of the cells. For example, old, unhealthy cells give smaller colonies, comparing with normal cells. So, like screening strains giving different size of colony, or presenting data showing KO cells giving smaller colony size, does not make a lot of sense to me. Am I wrong?
>   
you can not expect all colonies to be same size because we can not 
spread plate such that you have single cell in one spot.
> 5.      Chemotaxis: to show a mutant strain has chemotaxis defect, we would quantify the chemotaxis speed and directionality. However, chemotaxis is also dependent on how cells are polarized. For example, pi3k- cells would not show a lot of chemotaxis defect if they are pulsed long enough. The thing is "pulsed enough" or "polarized well" is an arbitrary concept. If polarizing is not paralleling, how meaningful to calculate the absolute number of "chemotaxis index"? At least we should not expect to get the same number from different labs, right?
>   
i am not an expert in this.
> 6.      Loading control of northern blot during development: to show a gene expression changes during development, usually we show an mRNA northern blot normalized to "total RNA". However, we all know that the cells size shrinks a lot during cells starvation and development, and the "total RNA" also decrease a lot in a cell. The real question in our mind actually is "how this gene changes in a cell during development", or "how this gene changes in a aggregate/slug/fruiting body during development". So I think normalization to total RNA is not accurate at all.  Should we normalize RNA-seq of dictyexpress to "cell number" instead of "total RNA"?
>   
i believe total RNA is the best way to normalize for northern blot.

-Deen
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